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The impact of the intestinal and fecal microbiome on animal health has received considerable attention in recent years and has direct implications for the veterinary and wildlife rehabilitation fields. To examine the effects of rehabilitation on the microbiome in Kemp’s ridley sea turtlesLepidochelys kempii, fecal samples from 30 incidentally captured juveniles were collected during rehabilitation. Samples were analyzed to determine alpha- (α) and beta- (β) diversity as well as the taxonomic abundance of the fecal microbiota during rehabilitation and in response to treatment with antibiotics. The fecal microbial communities of animals housed in rehabilitation for a ‘short-term’ stay (samples collected 0-9 d post-capture) were compared with ‘long-term’ (samples collected 10+ d post-capture) and ‘treated’ groups (samples collected from turtles that had received antibiotic medication). Results of this study indicate that the most dominant phylum in fecal samples wasBacteroidetes(relative abundance, 45.44 ± 5.92% [SD]), followed byFirmicutes(26.62 ± 1.58%),Fusobacteria(19.49 ± 9.07%), andProteobacteria(7.39 ± 1.84%). Similarly, at the family level,Fusobacteriaceae(28.36 ± 17.75%),Tannerellaceae(15.41 ± 10.50%),Bacteroidaceae(14.58 ± 8.48%), andRuminococcaceae(11.49 ± 3.47%) were the most abundant. Our results indicated that both antibiotic-treated and long-term rehabilitated turtles demonstrated a significant decrease in β-diversity when compared to short-term rehabilitated turtles. Our results likewise showed that the length of time turtles spent in rehabilitation was negatively correlated with α- and β-diversity. This study demonstrates the importance of a judicious use of antibiotics during the rehabilitation process and emphasizes the importance of limiting the length of hospital stays for sick and injured sea turtles as much as possible.

Off-bottom cultivation of oysters, Crassostrea virginica, is increasing in the Gulf of Mexico. The warm ambient air and water temperatures found in the Gulf of Mexico, coupled with the target market for off-bottom cultivated oysters for live raw consumption, raise concerns about the potential infections by human health pathogens, Vibrio parahaemolyticus and V. vulnificus. Regular practices associated with off-bottom cultivation, such as desiccation, expose oysters to ambient air to eliminate bio-fouling and are also known to increase these Vibrio spp. levels in oysters. Along with cultivation methods being introduced in the Gulf of Mexico, the use of triploid oysters is becoming increasingly popular. Triploid oysters are used a majority of the time in off-bottom cultivation due to their sterility, which results in rapid growth and high summer meat quality. Research also suggests that the lack of gonad tissue may correlate with lower Vibrio spp. levels in oysters. In this study, triploid and diploid oysters were cultured in Australian long line systems and subjected to two typical desiccation practices, air dried and freshwater dipped/air dried, and then evaluated for V. parahaemolyticus and V. vulnificus abundances over time. Three two-week long studies determined that Vibrio spp. levels in oysters that underwent either desiccation treatment returned to levels similar to those of submersed oysters by day three, referred to as returning to background levels. However, the Vibrio spp. levels in the treated oysters remained not significantly different from the elevated levels seen immediately following the desiccation treatment until seven days after re-submersion. There was no significant difference in Vibrio spp. levels between triploid and diploid oysters, nor a difference in the time of re-submersion needed to return levels to background. These results suggest that oysters that have been desiccated should be re-submersed for at least seven days prior to harvest to mitigate any human health risk contributed by desiccation practices, regardless of oyster ploidy.

We report the draft genome sequences of Cetobacterium somerae 2G Large and two Cetobacterium isolates, 2A and 8H, which may represent novel species. The isolates were recovered from the intestines of catfish, and the genomes will assist in research to understand their potential use as probiotics in aquaculture.

The expansion of off-bottom aquaculture to the Gulf of Mexico has raised public health concerns for human health officials. High temperatures in the Gulf of Mexico are associated with high levels of Vibrio parahaemolyticus and Vibrio vulnificus. Routine desiccation practices associated with off-bottom aquaculture expose oysters to ambient air, allowing Vibrio spp. to proliferate in the closed oyster. Currently, there is limited research on the length of time needed for Vibrio spp. levels in desiccated oysters to return to background levels, defined as the levels found in oysters that remain continually submersed and not exposed to ambient air. This study determined the time needed to return V. parahaemolyticus, V. vulnificus, and Vibrio cholerae levels to background levels in oysters exposed to the following desiccation practices: 3-h freshwater dip followed by 24-h ambient air exposure, 27-h ambient air exposure, and control. All oysters were submerged at least 2 weeks prior to the beginning of each trial, with the control samples remaining submerged for the duration of each trial. Vibrio spp. levels were enumerated from samples collected on days 0, 1, 2, 3, 7, 10, and 14 after resubmersion using a three-tube most-probable-number enrichment followed by BAX PCR. V. cholerae levels were frequently (92%) below the limit of detection at all times, so they were not statistically analyzed. V. parahaemolyticus and V. vulnificus levels in the 27-h ambient air exposure and the 3-h freshwater dip followed by 24-h ambient air exposure samples were significantly elevated compared with background samples. In most cases, the Vibrio spp. levels in oysters in both desiccation treatments remained elevated compared with background levels until 2 or 3 days post-resubmersion. However, there was one trial in which the Vibrio spp. levels did not return to background levels until day 7. The results of this study provide scientific support that oyster farmers should be required to implement a minimum 7-day resubmersion regimen. This length of time allowed the Vibrio spp. levels to become not significantly different across all treatments.

The objective of this study was to determine whether common bacterial catfish pathogens could attach and colonize surfaces commonly found in aquaculture facilities. In addition, we evaluated the role of calcium in biofilm formation. Attachment to polystyrene plates was used to quantify biofilm formation by five bacterial pathogens (i.e., Flavobacterium columnare, Aeromonas hydrophila, Edwardsiella ictaluri, E. tarda, and E. piscicida). Flavobacterium columnare and A. hydrophila formed thick biofilms that were enhanced by calcium supplementation. Biofilm formation was significantly lower in all Edwardsiella species tested and calcium had little to no effect on Edwardsiella biofilm formation. Attachment to natural and artificial surfaces was quantified by a standard plate count method. Scanning electron microscopy (SEM) was used to confirm biofilm formation on the substrates. Flavobacterium columnare formed biofilm on the liner, flexible PVC, and nets. Bamboo prevented F. columnare attachment and inhibited cell growth. Aeromonas hydrophila and E. ictaluri formed biofilm on all materials tested, although significant differences were found among substrates. While E. ictaluri failed to form biofilm on microtiter polystyrene plates, it was able to colonize and multiply on all aquaculture materials tested. Our results demonstrated that common bacterial pathogens had the potential of colonizing surfaces and may use biofilm as reservoirs in fish farms. Received July 19, 2016; accepted January 19, 2017.

Columnaris disease, caused by Flavobacterium columnare , is an emerging problem in the rainbow trout ( Oncorhynchus mykiss ) aquaculture industry of Idaho. All F. columnare isolates taken from disease outbreaks in the rainbow trout producing region of southern Idaho, and for rainbow trout, are all genomovar I. Virulence phenotyping of 11 genomovar I and 1 genomovar I/II isolates, taken from 6 different farms, suggests significant variation in virulence toward rainbow trout with the most virulent strains having an LD50 of 1 × 10 6 CFU/ml − 1 when using a waterborne challenge model. The least virulent strain required 8 × 10 6 CFU/ml − 1 to reach the LD50. AFLP fingerprinting of these strains and the resulting phylogenetic tree show that all of the strains tested had a percent similarity of 75% or higher, save one, the MS-FC-4 strain had F. columnare isolated from disease outbreaks in rainbow trout production have a higher degree of diversity than previously thought.

Background The equine conjunctival microbiota has often been reported to be dominated by Gram-positive species such as Staphylococcus sp., Bacillus sp., and Corynebacterium sp. However, traditional culture-based methods can only recover a fraction of the bacterial species present in the sample. Objectives This pilot study aimed at exploring the diversity of the equine conjunctival microbiota using culture-independent methods. Study design Eight horses were included in this study, and only eyes with normal ophthalmic examination (n = 15 eyes) were sampled. Methods Conjunctival biopsies (culture-independent) were collected, and DNA was extracted from the tissues. Bacterial communities in conjunctival biopsies were characterized by next-generation sequencing of the 16S rRNA genes. Individual reads were ascribed to operational taxonomic units (OTUs) using BLASTn and Greengenes databases. Species richness, evenness, and Good's coverage were determined for each conjunctiva-associated microbial community. Results Culture-independent samples produced a total of 329 bacterial OTUs. The main OTUs identified in the study belonged to the Gram-negative species Ralstonia mannitolilytica (88.0%), Nicoletella semolina (3.3%), and Pseudomonas tolaasii (1.5%). Conclusions Contrary to previously published data based on culture-dependent methods, the horse eye microbial community was dominated by Gram-negative bacteria of the phylum Proteobacteria.

Mucus of fish skin harbors complex bacterial com- munities that likely contribute to fish homeostasis. When the equilibrium between the host and its external bacterial symbi- onts is disrupted, bacterial diversity decreases while opportu- nistic pathogen prevalence increases, making the onset of path- ogenic bacterial infection more likely. Because of that relation- ship, documenting temporal and spatial microbial community changes may be predictive of fish health status. The 2010 Deepwater Horizon oil spill was a potential stressor to the Gulf of Mexico's coastal ecosystem. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to ana- lyze the bacterial communities (microbiome) associated with the skin and mucus of Gulf killifish (Fundulus grandis )t hat were collected from oiled and non-oiled salt marsh sites in Barataria Bay, LA. Water samples and fin clips were collected to examine microbiome structure. The microbiome of Gulf killifish was significantly different from that of the surrounding water, mainly attributable to shifts in abundances of Cyanobacteria and Proteobacteria. The Gulf killifish's microbiome was dominated by Gammaproteobacteria, specifi- cally members of Pseudomonas. No significant difference was found between microbiomes of fish collected from oiled and non-oiled sites suggesting little impact of oil contamination on fish bacterial assemblages. Conversely, seasonality significant- ly influenced microbiome structure. Overall, the high similarity observed between the microbiomes of individual fish observed during this study posits that skin and mucus of Gulf killifish have a resilient core microbiome.

The acquisition of gut microbes does not occur randomly and is highly dependent on host factors, environmental cues, and self-assembly rules exerted by the microbes themselves. The main objective of this project was to characterize how the gut microbiome develops during the early life stages of Channel Catfish and to identify i) which bacteria are the main constituents of the gut microbiome at different ontogenesis stages, and ii) at which time point(s) the gut microbiome stabilizes. High-throughput Illumina Miseq DNA sequencing of the V4 domain of the 16S rRNA gene was used to assess the microbial community composition during the life stages of Channel Catfish along with water and feed samples. Microbiomes from fertilized eggs, sac fry, swim up fry, pre-fingerlings, and fingerlings were all significantly distinct. OTUs analyses showed that the phylum Proteobacteria, Firmicutes, Fusobacteria and Cyanobacteria dominated the Channel Catfish gut microbiome. During the early stages of ontogenesis, the fish microbiome was dynamic and highly diverse, with significant shifts occurring between fertilized eggs to sac fry (6dph), and from sac fry to swim up fry (15dph). The gut microbiome stabilized between the pre-fingerlings and fingerlings stage (≤90dph) with an observed reduction in species richness. Feed had a more significantly contribution to the microbial colonization of the gut than water. We have identified the period in which the gut microbiome changes rapidly from 15dph until 21dph before stabilizing after 90dph.

A recently developed attenuated vaccine for Flavobacterium columnare has been demonstrated to provide superior protection for channel catfish, Ictalurus punctatus, against genetically diverse columnaris isolates. We were interested in examining the mechanisms of this protection by comparing transcriptional responses to F. columnare challenge in vaccinated and unvaccinated juvenile catfish. Accordingly, 58 day old fingerling catfish (28 days post-vaccination or unvaccinated control) were challenged with a highly virulent F. columnare isolate (BGSF-27) and gill tissues collected pre-challenge (0 h), and 1 h and 2 h post infection, time points previously demonstrated to be critical in early host-pathogen interactions. Following RNA-sequencing and transcriptome assembly, differential expression (DE) analysis within and between treatments revealed several patterns and pathways potentially underlying improved survival of vaccinated fish. Most striking was a pattern of dramatically higher basal expression of an array of neuropeptides (e.g. somatostatin), hormones, complement factors, and proteases at 0 h in vaccinated fish. Previous studies indicate these are likely the preformed mediators of neuroendocrine cells and/or eosinophilic granular (mast-like) cells within the fish gill. Following challenge, these elements fell to almost undetectable levels (>100-fold downregulated) by 1 h in vaccinated fish, suggesting their rapid release and/or cessation of synthesis following degranulation. Concomitantly, levels of pro-inflammatory cytokines (IL-1b, IL-8, IL-17) were induced in unvaccinated fish. In contrast, in vaccinated catfish, we observed widespread induction of genes needed for collagen deposition and tissue remodeling. Taken together, our results indicate an important component of vaccine protection in fish mucosal tissues may be the sensitization, proliferation and arming of resident secretory cells in the period between primary and secondary challenge.

In the southeastern USA, columnaris disease (caused by Flavobacterium columnare) typically affects catfish raised in earthen ponds from early spring until late summer. Recently, unusually severe outbreaks of columnaris disease occurred at the E. W. Shell Fisheries Center located in Auburn, AL, USA. During these outbreaks, catfish and other aquaculture and sport fish species that were in ponds located within the same watershed were affected. Our objective was to investigate the genetic diversity among F. columnare isolates recovered from different sites, sources, and dates to clarify the origin of these outbreaks and, ultimately, to better understand the epidemiology of columnaris disease. A total of 102 F. columnare isolates were recovered from catfishes (channel catfish Ictalurus puntactus, blue catfish I. furcatus, and their hybrid), bluegill Lepomis microchirus, Nile tilapia Oreochromis niloticus, largemouth bass Micropterus salmoides, egg masses, and water during columnaris outbreaks (from spring 2010 to summer 2012). Putative F. columnare colonies were identified following standard protocols. All isolates were ascribed to Genomovar II following restriction fragment length polymorphism analysis of the 16S rRNA gene. Genetic variability among the isolates was revealed by amplified fragment length polymorphism. Date of isolation explained most of the variability among our isolates, while host was the least influential parameter, denoting a lack of host specificity within Genomovar II isolates. The susceptibility of each of the isolates against commonly used antibiotics was tested by antibiogram. Our data showed that 19.6 and 12.7% of the isolates were resistant to oxytetracycline and kanamycin, respectively.

Hypoxia inducible factors (HIFs) are considered to be the master switch of oxygen-dependent gene expression with mammalian species. In most cases, regulation of HIF has been believed at posttranslational levels. However, little is known of HIF regulation in channel catfish, a species highly tolerant to low oxygen condition. Here we report the identification and characterization of HIP-1 alpha, HIF-2 alpha a, HIF-2 alpha b, HIF-3 alpha, and FIH-1 genes, and their mRNA expression under hypoxia conditions. The transcripts of the five genes were found to be regulated temporally and spatially after low oxygen challenge, suggesting regulation of HIF-alpha genes at pre-translational levels. In most tissues, the HIF-alpha mRNAs were down-regulated 15 h but up-regulated 5 h after hypoxia treatment. Of these HIP-alpha mRNAs, the expression of HIF-3 alpha mRNA was induced in the most dramatic fashion, both in the speed of induction and the extent of induction, compared to HIF-1 alpha and HIF-2 alpha genes, suggesting its importance in responses to hypoxia. (C) 2013 Elsevier Inc. All rights reserved.

Since the Deepwater Horizon oil spill in 2010, anecdotal observations of Red Snapper Lutjanus campechanus from the northern Gulf of Mexico exhibiting unusual external lesions have been reported. Two opportunistic bacterial fish pathogens, Vibrio vulnificus and Photobacterium damselae, were recovered from the fish and were deemed responsible for the abnormalities. However, the culturable microbiota of healthy Red Snapper has not yet been characterized. We analyzed the heterotrophic bacteria associated with healthy Red Snapper caught off the Louisiana coast. In total, 179 isolates from 60 fish were recovered from skin and mucus, and 43 isolates were obtained from anterior kidney. All isolates were identified by 16S ribosomal RNA gene sequencing. The Proteobacteria was the predominant phylum in both external and internal samples, followed by the Firmicutes and the Actinobacteria. Within the Proteobacteria, most isolates were members of the genera Vibrio and Photobacterium, and V. natriegens and P. damselae were the predominant species. The results of this study suggest that both Vibrio spp. and Photobacterium spp. are associated with the normal microbiota of healthy Red Snapper. Thus, the opportunistic fish pathogens recovered in previous studies cannot be deemed lesion-forming until Koch's postulates are fulfilled.

The selective effect that refrigeration exerts on the autochthonous microbiota of the eastern oyster was investigated using culture-independent methods. Oysters from two different locations along the Gulf Coast of Mexico were analyzed concurrently during 2-week refrigeration. Ribosomal intergenic sequence analysis (RISA) generated complex bacterial community fingerprints that provided a strong association between RISA-defined clusters and sample date. Denaturing gradient gel electrophoresis (DGGE) profiles obtained with universal primers did not reflect any association based on date or geographic origin of the samples. During the storage period, Vibrio parahaemolyticus was replaced, at least in overall abundance, with other nonpathogenic Vibrio species better adapted to cold temperatures. This study shows that refrigeration itself does not limit the growth of the bacterial community nor reduces its richness.

Flavobacterium columnare, the causative agent of columnaris disease, is a highly diverse species comprised by three genomovars. Genomovar II strains are more virulent toward catfishes than genomovar I isolates. The objective of this study was to compare the vaccine efficacy of avirulent mutants derived from genomovars I and II using a rifampicin-resistance strategy. First, we compared the efficacy of 13 genomovar II mutants in channel catfish (Ictalurus punctatus) fingerlings and identified mutant 17-23 as the best vaccine candidate based on their relative percent survival (RPS) against a highly virulent genomovar II strain (BGFS-27). In the second experiment, we vaccinated zebrafish (Danio rerio) with two genomovar II mutants (17-23 and 16-534) and FCRR (genomovar I mutant) followed by exposure to BGFS-27 strain. RPS values were 28.4, 20.3 and 8.1% for 17-23, 16-534, and FCRR, respectively. For experiments 3 and 4, we tested both 17-23 and FCRR in channel catfish fry and Nile tilapia (Oreochromis niloticus). In both experiments, vaccinated fish were divided in two groups and each challenged with either a genomovar I (ARS-1) or a II (BGFS-27) strain. Channel catfish fry vaccinated with 17-23 and FCRR followed by challenge with BGFS-27 resulted in RPS values of 37.0% and 4.4%. When fish were challenged with ARS-1, RPS values were 90.9% and 72.7% for fish vaccinated with 17-23 and FCRR, respectively. Nile tilapia vaccinated with 17-23 and FCRR followed by challenged with BGFS-27 had RPS values of 82.1% and 16.1%, respectively. When fish were challenged with strain ARS-1, RPS values were 86.9% and 75.5%. Overall, our results demonstrated that vaccination with genomovar II mutant 17-23 confers better protection in channel catfish and Nile tilapia than FCRR against columnaris disease caused by genomovar II. Both mutants were equally protective against columnaris caused by genomovar I showing that 17-23 mutant cross-protected against both genomovars.

Background The microbiota plays an essential role in host health, particularly through competition with opportunistic pathogens. Changes in total bacterial load and microbiota structure can indicate early stages of disease, and information on the composition of bacterial communities is essential to understanding fish health. Although Red Snapper (Lutjanus campechanus) is an economically important species in recreational fisheries and a primary aquaculture candidate, no information is available on the microbial communities of this species. The aim of this study was to survey the microbiota of apparently healthy, wild-caught Red Snapper from the Gulf of Mexico. Sampled Red Snapper showed no physical signs of disease. Tissues that are either primary entry routes for pathogens (feces, gill) or essential to disease diagnosis (blood) were sampled. Bacteria were enumerated using culture-based techniques and characterized by pyrosequencing. Results Aerobic counts of feces and gill samples were 107 and 104 CFU g-1, respectively. All individuals had positive blood cultures with counts up to 23 CFU g-1. Gammaproteobacteria dominated the microbiota of all sample types, including the genera Pseudoalteromonas and Photobacterium in feces and Pseudomonas in blood and gill. Gill samples were also dominated by Vibrio while blood samples had high abundances of Nevskia. High variability in microbiota composition was observed between individuals, with percent differences in community composition ranging from 6 to 76 % in feces, 10 to 58 % in gill, and 52 to 64 % in blood. Conclusions This study provides the first characterization of the microbiota of the economically significant Red Snapper via pyrosequencing. Its role in fish health highlights the importance of understanding microbiota composition for future work on disease prevention using microbial manipulation.

We present experimental data on susceptibility to columnaris disease, caused by the bacterium Flavobacterium columnare, in hybrid catfish (female channel catfish Ictalurus puncta- tus × male blue catfish I. furcatus) (C×B). Under our experimental conditions, C×B hybrids were significantly more resistant to columnaris disease caused by the highly virulent strain of F. columnare BGFS-27 (genomovar II) than channel catfish and blue catfish. Channel and blue catfish cumulative mortalities after immersion challenge were 74 and 87%, respectively, whereas mortality in the C×B hybrid was 31%. Susceptibility to the strain ARS-1 (genomovar I) was lower among all catfishes, although channel catfish was the least resistant species at 32% cumulative mortality. By contrast, C×B hybrid and blue catfishes were strongly resistant to the ARS-1 strain, with

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 104 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.

Infrapopulation genetic variation of the oioxenous, hermaphroditic flatworm Nasicola klawei (Monogenea: Capsalidae) infecting the nasal cavities of nine yellowfin tuna, Thunnus albacares , from the Gulf of Mexico was analyzed using the first internal transcribed spacer (ITS1) single strand conformation polymorphism (SSCP), ITS1 sequencing, and amplified fragment length polymorphism (AFLP). Of a total of 32 worms, six had unique ITS1-SSCP types and the rest was grouped by three types. Two worms of the same infrapopulation shared an ITS1-SSCP type in nine instances but no infrapopulation was monophyletic by ITS1-SSCP analysis. ITS1 sequences (420 bp) varied by 1–11 (0.2–2.6%) nucleotides. Twenty-three AFLP profiles of 80–110 bands failed to support genomic monophyly of any N . klawei infrapopulation. 28S rDNA (990 bp) sequences from four worms representing four infrapopulations were identical and matched conspecific GenBank sequences. Concordant ITS1-SSCP and AFLP analyses indicated that these N . klawei infrapopulations principally resulted from tuna being repeatedly colonized by planktonic, infective larvae (oncomiracidia) rather than by a single host colonization followed by parasite maturation, self-fertilization, and production of auto-infecting progeny.

Laboratory-reared, specific pathogen-free fingerling channel catfish, Ictalurus punctatus, and adult zebrafish, Danio rerio, were each separately exposed by immersion challenge to the etiological agent of “columnaris disease,”Flavobacterium columnare (Japanese Collection of Microorganisms 21327 strain). At 24-h post-immersion, fish exhibiting a “saddleback” lesion were fixed whole in 10% neutral buffered formalin. Skin samples approximately 5 mm2 were excised from both the margin and center of each saddleback lesion as well as from corresponding sites in control, non-challenged, fish before being prepared routinely for scanning electron microscopy (SEM). Skin samples from control channel catfish and zebrafish had uniform, contiguous epidermal cells with continuous or closely apposed cell margins and well-defined microridges. Channel catfish skin lesion samples had margins typified by epidermal sloughing and lesion centers that exhibited a multitude of rod-shaped bacterial cells, approximately 3–10 µm long × 0.3–0.5 µm wide, intermingled with cellular debris across a surface characterized by denuded, strongly ridged, or folded dermal connective tissue. Zebrafish skin lesion samples had a multitude of rod-shaped bacterial cells and exhibited comparable ultrastructural changes but some lacked scales. These findings are the first published SEM observations of columnaris disease and saddleback lesions in channel catfish and zebrafish and thereby advance our understanding of the ultrastructural characteristics of acute-stage saddleback lesions and columnaris disease pathogenesis.

Soil bacterial communities have significant influence on soilborne plant pathogens and, thus, crop health. The present study focuses on ribotyping soil bacterial communities in different peanut-cropping sequences in Alabama. The objective was to identify changes in microbial assemblages in response to cropping sequences that can play a role in managing soilborne plant pathogens in peanut. Four peanut-cropping sequences were sampled at the Wiregrass Research Station, Headland, AL in 2006 and 2007, including continuous peanut, 4 years of bahiagrass followed by peanut, peanut-cotton, and peanut-corn-cotton. Soil sampling was done at early and mid-season and at harvest. Bacterial community structure was assessed using ribosomal intergenic spacer analysis (RISA) combined with 16S rRNA cloning and sequencing. RISA results indicated >70% dissimilarities among different cropping sequences. However, 90% similarities were noticed among replicated plots of the same cropping sequences. Cropping sequences and time of soil sampling had considerable effect on soil microbial community structure. Bahiagrass rotation with peanut was found to have the highest bacterial diversity, as indicated by a high Shannon Weaver Diversity index. Overall, higher bacterial diversity was observed with bahiagrass and corn rotations compared with continuous peanut. The bacterial divisions Proteobacteria, Acidobacteria, Firmicutes, Bacteroidetes, and Actinomycetes were the predominant bacterial phyla found in all peanut-cropping sequences. The Proteobacteria taxa in these soils were negatively correlated with the abundance of members of division Firmicutes but, conversely, had a significant positive correlation with Gemmatimonadetes taxa. The prevalence of the division Actinomycetes was negatively correlated with the relative abundance of members of division Verrucomicrobia. These results indicate complex interactions among soil bacteria that are important contributors to crop health.

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 × 103 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 × 106 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.

Recovery of bacteria from infected fish during population sampling can be affected by factors including the type of assay, method of specimen preservation and concentration of bacteria present. Consequently, before use in field sampling, methods should be validated. The three objectives of this study were, first, to determine whether a channel catfish Ictalurus punctatus (Rafinesque) fingerling classified as positive for Gram-negative Edwardsiella ictaluri infection according to bacterial culture before freezing was also classified as positive after freezing, second, to determine how direct culture from the kidney (DIRECT), culture of homogenate (HOMOG) and standard PCR (PCR) agree with bacterial culture in terms of classifying fish as positive or negative and third, to estimate diagnostic sensitivity (dSe) and diagnostic specificity (dSp) for DIRECT, HOMOG and PCR. In fresh and frozen fish, as bacterial concentration decreased, the ability of each assay to detect positive fish also decreased, especially when there were

Vaccination is an important disease management strategy used to maintain human and animal health worldwide. Vaccines developed for aquaculture have reduced antibiotic use in fish production. Original fish vaccines were bacterins (formalin-killed bacteria) delivered through immersion or injection that induced humoral (antibody) immunity. Next generation vaccines relied on multiple killed antigens delivered with an adjuvant to enhance vaccine effectiveness. Work in the 1990s showed the use of various strategies to develop modified live vaccines for use in fish. A modified live vaccine is a live pathogen that has been rendered non-pathogenic or avirulent by physical, chemical, or genetic engineering methods. The modified live vaccine typically retains its ability to infect the host which allows for effective presentation of protective antigens to generate cellular immunity (CD4 or CD8 T-cell responses). Modified live vaccines are advantageous in that they can be easily delivered (i.e., by immersion to young fish) and stimulate both humoral and cellular immunity of long duration. Disadvantages include issues with modified live vaccine safety to the host and environment. A successful modified live vaccine for use in warm water aquaculture is used to highlight the live vaccine strategy.

Analysis of bacterial communities present in high-pressure (HP)-treated, quick-frozen (QF), and raw oysters was carried out during three different seasons. Bacterial numbers and species diversity in each sample were determined at 0, 7, 14, and 21 days of storage. Results showed that numbers of total aerobic bacterial counts (TABC) in treated oysters were significantly lower than in untreated oysters at day 0 by 10 to 10(5) colony forming units per gram of oyster meat (CFU/g) in all samplings. However, an increase in TABC in HP-treated oysters was observed at days 7, 14, and 21 indicating that some bacteria survived the treatment and were able to proliferate during refrigeration conditions. Surprisingly, TABC in HP-treated oysters reached 10(8) CFU/g at 14 days of storage in all samplings (higher than TABC from raw oysters in two of the samplings performed). Analysis of the bacterial flora by 16S rDNA sequencing, revealed six different classes within the bacterial communities. The majority were Gram-negative bacteria, with the Gammaproteobacteria class representing between 56% and 92%. The most common bacterial genera found in this study were Shewanella, Vibrio and Psychrobacter. Four species of human pathogenic bacteria were also identified: V. vulnificus, V. parahaemolyticus, V. alginolyticus, and A. hydrophila although V. vulnificus was detected only in raw oysters.

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 (P0.05) 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 (P0.05) 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.

Comparison of the 23S rRNA gene sequences of Edwardsiella tarda and Edw. ictaluri confirmed a close phylogenetic relationship between these two fish pathogen species and a distant relation with the 'core' members of the Enterobacteriaceae family. Analysis of the rrl gene for 23S rRNA in Edw. ictaluri revealed the presence of an intervening sequence (IVS) in helix-45. This new 98bp IVS shared 97% nucleotide identity with Salmonella typhimurium helix-45 IVS. Edw. ictaluri helix-45 IVS was present in all Edw. ictaluri strains analyzed and in at least six rrl operons within each cell. Fragmentation of 23S rRNA due to IVS excision by RNase III was observed by methylene blue staining of ribosomal RNA extracted from Edw. ictaluri isolates. This is the first report of an IVS in the 23S rRNA gene of the genus Edwardsiella.

International audience; The external microbiome of fish is thought to benefit the host by hindering the invasion of opportunistic pathogens and/or stimulating the immune system. Disruption of those microbial communities could increase susceptibility to diseases. Traditional aquaculture practices include the use of potent surface-acting disinfectants such as potassium permanganate (PP, KMnO4) to treat external infections. This study evaluated the effect of PP on the external microbiome of channel catfish and investigated if dysbiosis leads to an increase in disease susceptibility. Columnaris disease, caused by Flavobacterium columnare, was used as disease model. Four treatments were compared in the study: (I) negative control (not treated with PP nor challenged with F. columnare), (II) treated but not challenged, (III) not treated but challenged, and (IV) treated and challenged. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to analyze changes in the external microbiome during the experiment. Exposure to PP significantly disturbed the external microbiomes and increased catfish mortality following the experimental challenge. Analysis of similarities of RISA profiles showed statistically significant changes in the skin and gill microbiomes based on treatment and sampling time. Characterization of the microbiomes using 16S rRNA gene pyrosequencing confirmed the disruption of the skin microbiome by PP at different phylogenetic levels. Loss of diversity occurred during the study, even in the control group, but was more noticeable in fish subjected to PP than in those challenged with F. columnare. Fish treated with PP and challenged with the pathogen exhibited the least diverse microbiome at the end of the study.

This study investigated the impacts of bacterial DNA extraction methodology on downstream analysis of fish gut microbiota. Feces and intestine samples were taken from three sympatric freshwater fish species with varying diets. Samples were processed immediately (approximately 4 h after capture; fresh), stored at -20 °C for 15 days or preserved in RNAlater® reagent for 15 days. DNA was then extracted using two commercial kits: one designed for animal tissues and one specifically formulated for stool samples. Microbial community fingerprints were generated using ribosomal intergenic spacer analysis. Factors including diversity as depicted by band number, band intensity, repeatability and practicalities such as cost and time were considered. Despite significant differences in microbiota structure, results were similar between feces and intestine samples. Frozen samples were consistently outperformed by other storage methods and the stool kit typically outperformed the tissue kit. Overall, we recommend extraction of bacterial DNA from fresh samples using the stool kit for both sample types. If samples cannot be processed immediately, preservation in RNAlater® is preferred to freezing. Choice of DNA extraction method significantly influences the results of downstream microbial community analysis and thus should be taken into consideration for metadata analysis.

Intra- and interspecific characteristics of fish-pathogenic Edwardsiella ictaluri, and E. tarda were determined by numerical analysis of gel electrophoresed protein profiles, fatty acid methyl esters (FAMEs) and immunoblotting. The 18 E. ictaluri isolates revealed a high degree of homogeneity (70% similarity or higher) in their protein profiles and 95% similarity in their FAME, while the nine E. tarda isolates revealed 30% similarity in their protein profiles and 95% similarity in their FAME. Immunoblots probed for antigenic epitopes with goat antiserum produced against E. ictaluri and E. tarda, respectively, revealed that E. ictaluri were more homogeneous compared with the E. tarda isolates. Overall, there was a considerable degree of relatedness between the two species. Our findings suggest that phenotypically E. ictaluri represents a clonal bacterial population structure compared with the less monomorphic E. tarda.

A specific and rapid PCR detection method for Flavobacterium columnare based on the 16S-23S rDNA intergenic spacer region (ISR) of the ribosomal RNA operon has been developed. The ISR of 30 F. columnare strains and other Flavobacterium species was amplified using universal primers and sequenced. Once F. columnare specific sequences within the ISR were recognized, spe- cific PCR primers were designed against them (FCISRFL and FCISRR1). The primers were sensitive and able to detect as low as 7 colony forming units from pure culture by PCR. The new PCR detection method was applied to experimentally infected channel catfish. Two different experiments in which channel catfish fingerlings were infected by intramuscular injection or by immersion bath showed the advantage of the PCR method over standard culture techniques. F. columnare was detected by PCR in both tank water and catfish tissue samples with a higher frequency and in less time than standard microbiological methods. Furthermore, PCR detection confirmed that F. columnare can be transmit- ted horizontally indirectly through the water column without fish-to-fish contact. The newly devel- oped PCR detection method for F. columnare was more sensitive and rapid than standard culture on bacteriological media for detection of F. columnare in channel catfish tissues and in tank water.

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.

Coronatine is a polyketide phytotoxin produced by several plant pathogenic Pseudomonas spp. The effect of coronatine on abscission in Citrus sinensis L. Osbeck 'Hamlinʼ and 'Valenciaʼ orange fruit, leaves, fruitlets, and fl owers was determined. Coronatine at 200 mg·L-1 signifi cantly reduced fruit detachment force of mature fruit, and did not cause fruitlet or fl ower loss in 'Valenciaʼ. Cumulative leaf loss was 18% with coronatine treatment. Coronafacic acid or coronamic acid, precursors to coronatine in Pseudomonas syringae, did not cause mature fruit abscission. Ethylene production in mature fruit and leaves was stimulated by coronatine treatment, and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and 12-oxo-phytodienoate reductase (12-oxo-PDAR) gene expression was upregulated. A slight chlorosis developed in the canopy of whole trees sprayed with coronatine, and chlorophyll content was reduced rela- tive to adjuvant-treated controls. Leaves formed after coronatine application were not chlorotic and had chlorophyll contents similar to controls. Comparison of coronatine to the abscission compounds methyl jasmonate, 5-chloro-3- methyl-4-nitro-pyrazole and ethephon indicated differences in ethylene production and ACO and 12-oxo-PDAR gene expression between treatments. Leaf loss, chlorophyll reduction and low coronatine yield during fermentation must be overcome for coronatine to be seriously considered as an abscission material for citrus. Coronatine is a polyketide phytotoxin produced by several plant pathogenic Pseudomonas spp. (Mitchell 1982; Volksch and Wein- gart 1998). In compatible host-pathogen interactions, coronatine was associated with virulence and symptom development. Coro- natine induces a diffuse nonhost-specifi c chlorosis after application

A putative thermostable pectinmethylesterase (TSPME) protein of 36 kDa was isolated from heat-treated citrus finisher pulp. After purification and partial sequencing of the protein, a reverse genetic approach was used to obtain the complete genomic sequence of a new pectinmethylesterase (PME) gene, CsPME4, from Citrus sinensis (L.) Osb. cv. Valencia. The CsPME4 gene contained two exons of 1203 and 690 bp interrupted by a single positionally conserved intron of 1230 bp. A full-length CsPME4 cDNA clone amplified from Valencia orange juice vesicles shared 98% identity with the genomic clone. The encoded protein of the full-length CsPME4 cDNA shared 66 and 39% amino acid identity with the full-length encoded proteins of the citrus PME, CsPME1, and CsPME3, respectively, whereas the predicted mature protein of CsPME4 shared 80 and 61% identity with the predicted mature proteins of CsPME1 and CsPME3, respectively. Southern analysis demonstrated that CsPME4 was present in at least two copies in the Valencia orange genome. Northern analysis revealed that CsPME4 mRNA was accumulated mainly in young and developing tissues of Valencia orange. Several approaches to express recombinant CsPME4 in different systems failed to obtain active protein. Further research will be necessary to successfully express the putative TSPME gene CsPME4 for biochemical characterization.

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.

Summary A polyphasic taxonomic approach using phenotypic and molecular genetic techniques, was carried out on the species Vibrio vulnificus in order to study its intraspecific diversity. Seven techniques, including phenotypic (API 20E, BIOLOG, total protein profiles, serotyping, ELISA), and genotypic methods (ribotyping and AFLP), were employed on 80 V. vulnificus strains of biotypes 1 and 2, including 9 reference cultures. The isolates came from different geographic origins (USA, Spain, Belgium, Sweden, Norway, Japan, Thailand) and types of samples (clinical, health/diseased fish, seafood, water). Diversity indexes calculated for strains of both biotypes revealed a higher phenotypic and genetic diversity of biotype 1 strains in contrast with the homogeneity shown by biotype 2 strains. A species profile was established for the species V. vulnificus by BIOLOG and SDS-PAGE. Serotyping allowed a clear separation within species level, and grouped typical and atypical biotype 2 strains in one serovar. Genetic fingerprinting techniques, especially AFLP, were able to differentiate within strains belonging to the same serovar, allowing the tracking of specific clones in epidemiologic or ecological studies. The techniques employed do not support the maintenance of the two biotypes established for the 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.

Flavobacterium columnare is a bacterial fish pathogen that affects many freshwater species worldwide. The natural reservoir of this pathogen is unknown, but its resilience in closed aquaculture systems posits biofilm as the source of contagion for farmed fish. The objectives of this study were (i) to characterize the dynamics of biofilm formation and morphology under static and flow conditions and (ii) to evaluate the effects of temperature, pH, salinity, hardness, and carbohydrates on biofilm formation. Nineteen F. columnare strains, including representatives of all of the defined genetic groups (genomovars), were compared in this study. The structure of biofilm was characterized by light microscopy, confocal laser scanning microscopy, and scanning electron microscopy. F. columnare was able to attach to and colonize inert surfaces by producing biofilm. Surface colonization started within 6 h postinoculation, and microcolonies were observed within 24 h. Extracellular polysaccharide substances and water channels were observed in mature biofilms (24 to 48 h). A similar time course was observed when F. columnare formed biofilm in microfluidic chambers under flow conditions. The virulence potential of biofilm was confirmed by cutaneous inoculation of channel catfish fingerlings with mature biofilm. Several physicochemical parameters modulate attachment to surfaces, with the largest influence being exerted by hardness, salinity, and the presence of mannose. Maintenance of hardness and salinity values within certain ranges could prevent biofilm formation by F. columnare in aquaculture systems.

The prevalence of Vibrio vulnificus on the external surfaces of fish from the northern Gulf of Mexico was determined in this study. A collection of 242 fish comprising 28 species was analyzed during the course of 12 sampling trips over a 16-month period. The prevalence of V. vulnificus was 37% but increased up to 69% in summer. A positive correlation was found between the percentages of V. vulnificus -positive fish and water temperatures, while salinity and V. vulnificus -positive fish prevalence were inversely correlated. A general lineal model (percent V. vulnificus -positive fish = 0.5930 − 0.02818 × salinity + 0.01406 × water temperature) was applied to best fit the data. Analysis of the population structure was carried out using 244 isolates recovered from fish. Ascription to 16S rRNA gene types indicated that 157 isolates were type A (62%), 72 (29%) were type B, and 22 (9%) were type AB. The percentage of type B isolates, considered to have greater virulence potential, was higher than that previously reported in oyster samples from the northern Gulf of Mexico. Amplified fragment length polymorphism (AFLP) was used to resolve the genetic diversity within the species. One hundred twenty-one unique AFLP profiles were found among all analyzed isolates, resulting in a calculated Simpson's index of diversity of 0.991. AFLP profiles were not grouped on the basis of collection date, fish species, temperature, or salinity, but isolates were clustered into two main groups that correlated precisely with 16S rRNA gene type. The population of V. vulnificus associated with fishes from the northern Gulf of Mexico is heterogeneous and includes strains of great virulence potential.

Depuration under different salinities was used to reduce the human pathogen Vibrio vulnificus from Eastern oysters (Crassostrea virginica). Individual recirculating systems were used to test the efficacy of depuration at three salinities (15, 25, and 35 psu) in four independent trials during a 14 day period. Initial loads of V. vulnificus were higher than 10,000 MPN/g of oyster meat in all trials. Data showed that 25 and 35 psu treatments were more efficient in reducing V. vulnificus numbers than 15 psu with an overall reduction of >3 logs. A significant decrease in MPN/g was observed as early as day 6 and further reductions were observed at day 10, while longer depurations did not improve efficacy. Only the highest salinity (35 psu) was capable of reducing V. vulnificus numbers to the FDA recommended level of

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill- adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10 6 colony forming units (CFU) g -1 ), but significant differences in adhe- sion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant differ- ence was found between both strains when adhered to skin, but adhered cell numbers were lower (10 3 CFU g -1 ) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (>10 6 CFU g -1 ), while only

The Deepwater Horizon Oil Spill was the largest oil spill in USA history releasing approximately 4.9 million barrels of crude oil into the Gulf of Mexico. Soon after the spill started, tar balls and other forms of weathered oil appeared in large numbers on beaches in Mississippi and Alabama. In this study, we analyzed tar balls for total aerobic bacterial (TAB) counts and also for the presence of Vibrio vulnificus, a human pathogen known to be abundant in the Gulf Coast environment and capable of causing severe wound infections by contact with contaminated surfaces. Our results showed that TAB counts were significantly higher in tar balls than in sand and seawater collected at the same location. In addition, V. vulnificus numbers were 10× higher in tar balls than in sand and up to 100× higher than in seawater. Densities of V. vulnificus were higher than 10(5) colony forming units/g of tar ball in all samples analyzed. Our data suggest that tar balls can act as reservoirs for bacteria including human pathogens.

Heterogeneity among ribosomal operons in Vibrio vulnificus is purported as a probabilistic indicator of strain virulence and classifies V. vulnificus strains as 16S rRNA genes type A and B. In this study, 16S rRNA genes typing of V. vulnificus strains isolated from the Valencia city coast, in the western Mediterranean, showed that 24 out of 30 isolates were type A, one was type B and five could not be typed. Single strand conformation polymorphism (SSCP) analysis of this gene region revealed complex patterns indicative of intragenomic ribosomal operon sequence heterogeneity. The 16S rRNA genes of three untypeable isolates C27, C30, and C34, along with type A (ATCC 27562) and B (C7184) reference strains, were amplified, cloned and sequenced. The number of unique 16S rRNA gene sequences was 4, 3, and 4 for the environmental isolates. The type strain of the species (ATCC 27562) presented only two 16S rRNA gene types, while the reference isolate C7184 of clinical origin had only one 16S rRNA gene type. Sequences differed from five to 35 bp (99.6% to 97.6% sequence similarity). Areas of variability concentrated in helices 10, 18, and 37 and included variants with short intervening sequences in helix 10. Most of the substitutions showed compensatory mutations suggesting ancient sequence divergence generated by lateral gene transfer.

The Flavobacterium columnare heat shock protein (HSP) gene dnaJ* was isolated, cloned, expressed, and used as an antigen in a recombinant vaccine strategy for channel catfish Ictalurus punctatus. The F. columnare dnaJ* sequence was obtained from genomovars I and II and showed intraspecies variability. Recombinant protein was expressed and purified from Escherichia coli cultures and injected intraperitoneally (12 microg of purified DnaJ/fish) into fingerling channel catfish. In addition, induced (expressing the recombinant DnaJ) and uninduced (no recombinant protein being produced) E. coli cultures were also used to immunize fish. At 28 d postimmunization, antibody response was evaluated and the fish were challenged with F. columnare. A specific immune response against DnaJ was observed in fish immunized with DnaJ or E. coli cultures expressing DnaJ. No protection against the disease, however, was observed in F. columnare-challenged fish that had been immunized with DnaJ. Some level of protection was observed in fish immunized with uninduced and induced E. coli lysates. Although HSPs have been shown to be immunodominant and good candidates for subunit vaccines in other animals, DnaJ failed to protect against columnaris disease in channel catfish.

The efficacy of a flow-through depuration system in eliminating the human pathogen Vibrio vulnificus from eastern oysters ( Crassostrea virginica ) collected from the North Gulf of Mexico coast was evaluated in this study. Depuration experiments were conducted with artificially inoculated oysters using laboratory-grown strains of V. vulnificus as well as with naturally contaminated oysters. Determination of V. vulnificus numbers in oyster tissues was conducted at 0, 1, 2, 3 and 6 days of depuration. Results showed that the depuration of V vulnificus is possible using a flow-through system. Numbers of V. vulnificus in laboratory-inoculated oysters were reduced from >100,000 Most Probable Number (MPN)/g of oyster tissue to 23 MPN/g after six days of depuration. As expected depuration results of naturally contaminated oysters were more variable. Depuration at low temperature (15°C) had very little success in reducing the numbers of V. vulnificus in oyster tissues. On the contrary, when flow rate was increased from 11 L/m to 68 L/m, numbers of V. vulnificus in oysters were reduced from a starting concentration of 110,000 MPN/g to 3 MPN/g in six days. Nevertheless, a high-flow rate was not enough to eliminate V. vulnificus from oysters consistently. Vibrio vulnificus was effectively eliminated from oysters only when incoming water salinity was higher than 30 parts per thousand (ppt). Depuration did not select for pathogenic V. vulnificus strains. Pre- and post depuration V. vulnificus isolates contained similar proportions of the proposed more virulent type.

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°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.

Three genes from a shotgun genomic library of Flavobacterium columnare virulent strain ALG-00-530 were identified, characterized, and selected for differential expression analysis based on sequence similarity to putative virulence genes from related species. These genes were: glycosyltransferase (gtf), nitric oxide reductase (norB) and thioredoxin (trx). A collection of 30 F. columnare strains, including strains from genomovars I and II, were tested for the presence of these genes. Distribution patterns of gtf, norB, and trx across the species were not uniform. Nucleotide sequence variation was observed between genomovars for each gene; however, strains within the same genomovar shared identical gene sequences. Nine strains of F. columnare, from both genomovars, were chosen for gene expression analysis. The expression profile of these genes varied when selected strains were grown in vitro under identical conditions. ALG-00-530, a high virulent strain, was chosen for gene expression comparison under standard growing conditions, iron-limited conditions and in the presence of skin explants from channel catfish. NorB, and trx gene expression levels varied when ALG- 00-530 was incubated under different conditions.

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.