Cova R. Arias

Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish

Aims:  To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare

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

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.

First Report of Yersinia ruckeri Biotype 2 in the USA

A polyphasic characterization of atypical isolates of Yersinia ruckeri (causative agent of enteric redmouth disease in trout) obtained from hatchery-reared brown trout Salmo trutta in South Carolina was performed. The Y. ruckeri isolates were biochemically and genetically distinct from reference cultures, including the type strain, but were unequivocally ascribed to the species Y. ruckeri, based on API 20E, VITEK, fatty acid methyl ester profiles, and 16S rRNA gene sequencing analysis. These isolates were nonmotile and unable to hydrolyze Tween 20/80 and were therefore classified as Y. ruckeri biotype 2. Genetic fingerprint typing of the isolates via enterobacterial repetitive intergenic consensus (amplified by polymerase chain reaction) and fragment length polymorphism showed biotype 2 as a homogeneous group distinguishable from other Y. ruckeri isolates. This is the first report of Y. ruckeri biotype 2 in the USA.

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.

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.

Isolation of Edwardsiella ictaluri from Tadpole Madtom in a Southwestern New Jersey River

Abstract In 2002, New Jersey Division of Fish and Wildlife biologists conducted an aquatic assessment in a section of the Cohansey River in Cumberland County. Three of seven tadpole madtoms Noturus gyrinus collected were observed to have an ulcerative lesion on the head between the eyes. Gram-negative, oxidase-negative rods were isolated from the head kidneys of two of the madtoms and from the cranial ulcer of the third madtom. Four different identification methods— API-20E, whole-cell fatty acid methyl esters (FAME), BIOLOG, and enterobacterial repetitive intergenic consensus (ERIC) sequences—ascribed the isolates to the species Edwardsiella ictaluri. A phenotypic comparison using FAME and BIOLOG data revealed that E. ictaluri isolates from wild madtoms shared a high degree of similarity to E. ictaluri isolates from cultured channel catfish with enteric septicemia of catfish (ESC) from southeastern states. However, fingerprinting by amplified fragment length polymorphisms (AFLP) demonstrated that the thr...

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.

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.

Evaluation of Global Gene Expression During Cold Shock in the Human Pathogen Vibrio vulnificus

Vibrio vulnificus can adapt to cold temperatures by changing the expression profiles of certain genes and their resulting proteins. In this study, the complete V. vulnificus transcriptome was analyzed under cold shock by looking at gene expression changes occurring during the shift from 35°C to 4°C. A DNA microarray-based global transcript profiling of V. vulnificus showed that 165 genes out of 4,488 altered their expression profiles by more than twofold. From 35°C to 10°C, an overall gene repression was observed while changes occurring below 10°C mainly resulted in upregulation. The highest induction observed occurred in two of the five categorized cold-shock genes, cspA and cspB, which showed a complementary expression pattern during cold shock suggesting a homologous role. Other genes showing a significant fold increase included ribosomal genes, protein folding regulators, and membrane genes. Repressions were observed in all orthologous groups. Genes with top fold changes in repression include those coding for catalytic enzymes responsible for non temperature-related stress regulation. These included antioxidants, sugar uptake, and amino acid scavengers. V. vulnificus maintained a high level of cspA and cspB transcripts during the entire experiment suggesting that these class I cold-shock genes are required beyond the initial phase of the acclimation period.

Comparative Phenotypic and Genotypic Analysis of Edwardsiella Isolates from Different Hosts and Geographic Origins, with Emphasis on Isolates Formerly Classified as E. tarda, and Evaluation of Diagnostic Methods

ABSTRACT Edwardsiella spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as Edwardsiella tarda actually represent three genetically distinct yet phenotypically ambiguous taxa with various degrees of pathogenicity in different hosts. Previous recognition of these taxa was hampered by the lack of a distinguishing phenotypic character. Commercial test panel configurations are relatively constant over time, and as new species are defined, appropriate discriminatory tests may not be present in current test panel arrangements. While phenobiochemical tests fail to discriminate between these taxa, data presented here revealed discriminatory peaks for each Edwardsiella species using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) methodology, suggesting that MALDI-TOF can offer rapid, reliable identification in line with current systematic classifications. Furthermore, a multiplex PCR assay was validated for rapid molecular differentiation of the Edwardsiella spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of Edwardsiella spp. and advantages of employing alternative single-copy genes gyrB and sodB for molecular identification and classification of Edwardsiella were demonstrated. Last, sodB sequencing confirmed that isolates previously defined as typical motile fish-pathogenic E. tarda are synonymous with Edwardsiella piscicida, while atypical nonmotile fish-pathogenic E. tarda isolates are equivalent to Edwardsiella anguillarum. Fish-nonpathogenic E. tarda isolates are consistent with E. tarda as it is currently defined. These analyses help deconvolute the scientific literature regarding these organisms and provide baseline information to better facilitate proper taxonomic assignment and minimize erroneous identifications of Edwardsiella isolates in clinical and research settings.