Aeromonas veronii is a gram-negative species abundant in aquatic environments that causes disease in humans as well as terrestrial and aquatic animals. In the current study, 41 publicly available A. ...veronii genomes were compared to investigate distribution of putative virulence genes, global dissemination of pathotypes, and potential mechanisms of virulence. The complete genome of A. veronii strain ML09-123 from an outbreak of motile aeromonas septicemia in farm-raised catfish in the southeastern United States was included. Dissemination of A. veronii strain types was discovered in dispersed geographical locations. Isolate ML09-123 is highly similar to Chinese isolate TH0426, suggesting the two strains have a common origin and may represent a pathotype impacting aquaculture in both countries. Virulence of strain ML09-123 in catfish in a dose-dependent manner was confirmed experimentally. Subsystem category disposition showed the majority of genomes exhibit similar distribution of genomic elements. The type I secretion system (T1SS), type II secretion system (T2SS), type 4 pilus (T4P), and flagellum core elements are conserved in all A. veronii genomes, whereas the type III secretion system (T3SS), type V secretion system (T5SS), type VI secretion system (T6SS), and tight adherence (TAD) system demonstrate variable dispersal. Distribution of mobile elements is dependent on host and geographic origin, suggesting this species has undergone considerable genetic exchange. The data presented here lends insight into the genomic variation of A. veronii and identifies a pathotype impacting aquaculture globally.
To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we ...sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo -inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo -inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo -inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.
In the United States, catfish are primarily farmed in earthen ponds, resulting in an aquatic environment influenced both by management practices and natural ecological processes. Profiling pond water ...microbiota can be useful for understanding what conditions may lead to microbial communities associated with production issues and could inform management practices. The aim of this study was to identify appropriate methods for bacterial community profiling of catfish pond water with nanopore sequencing of the 16S rRNA gene. To this end, two forward primers, two reverse primers, and three different polymerase chain reaction (PCR) cycle numbers were tested on a mock community consisting of aquaculture‐relevant bacteria. The optimized protocol was applied to water samples obtained from three experimental catfish ponds sampled in May, June, August, and September 2020. Applying these methods to pond samples allowed for the identification of 1488 genera, primarily within four dominating phyla: Actinobacteriota, Bacteroidota, Cyanobacteria, and Firmicutes. High variation was observed between individual ponds and sampling timepoints; only 18 of the 1488 genera were found in relative abundances ≥1% in all ponds from at least one sampling point. Despite this variation, consistent results could be observed. Samples obtained from the month of September had the highest number of observed genera, and samples from June had the lowest. Overall, these data demonstrated individual ponds represent distinct microcosms composed of unique bacterial communities, although this pond effect was secondary to the influence of sampling month on pond community composition.
Lineages of hypervirulent
(vAh) are the cause of persistent outbreaks of motile
septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of
has resulted in annual losses ...of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of
affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61
spp. genomes, of which 40 were affiliated with
, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with
. Within this lineage, conserved genetic loci unique to
were identified, with some genes present in consistently higher copy numbers than in non-epidemic
isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (
, and
) which may have functional implications. Collectively, these results characterize a hypervirulent
pathotype that affects farmed fish on multiple continents.
Streptococcus iniae is a re-emerging bacterial pathogen in freshwater and marine aquaculture worldwide. There are no commercial vaccines available for S. iniae in the United States, and autogenous ...vaccines are restricted to inactivated whole-cell preparations with limited protection against heterogenous strains. Live-attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they induce robust cellular and humoral immunity, and may provide longer lasting protection through less stressful routes of administration. We investigated whether accumulation of mutations in S. iniae by serial passage in the presence of rifampin can generate immunogenic LAV conferring protection against challenge with heterologous wild-type (WT) S. iniae strains in Nile tilapia (Oreochromis niloticus). Three lineages of rifampin-resistant S. iniae strains were generated from three genetically distinct parent strains (n = 9) by multiple passages in increments of Rifamycin SV sodium salt. Growth in liquid media, extent of capsulation, antimicrobial susceptibility, survival in Nile tilapia whole blood, and cytotoxicity in an O. mossambicus endothelial cell line were compared between the passaged and WT strains. Nile tilapia challenges were used to assess strain virulence, generation of anti-S. iniae IgM, and the protection conferred by LAV candidates against virulent S. iniae. Rifampin-resistant strains demonstrated changes in growth rate and cytotoxicity in endothelial cells, as well as significant reductions in whole blood survival (p < 0.05). Selected strains also showed attenuated virulence in the Nile tilapia challenge model, and anti-S. iniae IgM generated against these strains demonstrated cross-reactivity against heterologous bacteria. Immunization by intracoelomic injection induced protection against a virulent WT strain of S. iniae, with relative percent survival up to 95.05%.
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•Serial passaging in increasing concentrations of rifampin attenuated Streptococcus iniae.•Passaged strains produced specific and cross-reactive antibody responses in tilapia.•A strain offering high homologous protection has potential as a live vaccine.
Abstract
Bolbophorus damnificus
is a digenetic trematode causing significant economic losses within the United States commercial catfish industry. The indirect life cycle is complex, requiring ...piscivorous birds, aquatic snails, and fish to complete. With federal protections on many piscivorous birds and no FDA‐approved therapeutic treatment for trematode infections in fish, management within the catfish industry is primarily limited to controlling the snail host.
Planorbella trivolvis
and
Biomphalaria havanensis
are snails commonly found in commercial catfish ponds and known to transmit
B. damnificus
. This study evaluated a low‐cost snail trap prototype for monitoring populations of these snails in efforts to better inform treatment regimes. Snail traps were deployed into ponds on a commercial catfish farm and checked weekly throughout the production season. All snails were identified and counted to evaluate trap efficacy. Traps captured significantly more snails in foodfish ponds than in fingerling ponds; however, this trend was not present in monthly box samples.
B. havanensis
was rarely collected and was only found consistently within one pond, while
P. trivolvis
was routinely collected from all ponds, although abundance varied widely among ponds. Snail traps could be a useful, low‐cost method to monitor snail populations within ponds and better inform control treatments.
Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific ...variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG5) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G+C content demonstrated 56.4% G+C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.
Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. ...piscicida–like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published primers, for E. tarda, E. piscicida, and E. piscicida–like sp. to provide rapid quantitative confirmatory tests for these phenotypically ambiguous bacteria. The qPCR assays were shown to be repeatable and reproducible, with high degrees of sensitivity and specificity. Each assay showed a linear dynamic range covering 8 orders of magnitude and a sensitivity limit of 5 copies of target DNA in a 15-µL reaction. In addition, each assay was found specific to their respective targets with no observed amplification from nontarget organisms, including the closely related E. ictaluri and E. hoshinae. Under the conditions used in this study, the 3 assays had a quantifiable limit ranging from 103 (E. piscicida) to 102 (E. piscicida–like and E. tarda) colony forming units in kidney tissue biopsies (approximately 25 mg), pond water samples (35 mL), and broth culture (20 μL). In experimental challenges, the assays were able to detect their respective targets in both clinically and subclinically infected channel catfish (Ictalurus punctatus) fingerlings. In addition to quantifying target bacteria from various substrates, the assays provide rapid identification, differentiation, and confirmation of the phenotypically indistinguishable E. tarda, E. piscicida, and E. piscicida–like sp., a valuable tool for diagnostic assessments.
spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as
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
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
spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of
spp. and advantages of employing alternative single-copy genes
and
for molecular identification and classification of
were demonstrated. Last,
sequencing confirmed that isolates previously defined as typical motile fish-pathogenic
are synonymous with
, while atypical nonmotile fish-pathogenic
isolates are equivalent to
Fish-nonpathogenic
isolates are consistent with
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
isolates in clinical and research settings.
The commercial channel catfish (Ictalurus punctatus) industry is adversely impacted by enteric septicemia of catfish (ESC), caused by the Gram‐negative enteric rod Edwardsiella ictaluri. In efforts ...to develop more effective management strategies, the use of a live‐attenuated E. ictaluri isolate as an oral vaccine was investigated. The utility and broad application of a vaccine is dependent on the protection offered against multiple field isolates. Immunization trials, followed by bacterial challenge with archived virulent E. ictaluri isolates from the catfish farming region of the southeastern United States, were conducted over a 3‐year period. These isolates were archived from 1993 to 2016 and included representatives from nonvaccinated and vaccinated populations of diseased channel catfish in Mississippi, an isolate from nonvaccinated hybrid catfish raised in Mississippi, and an isolate from nonvaccinated channel catfish from Alabama. Molecular characterization of the isolates revealed a high degree of genetic similarity evidenced by repetitive sequence‐mediated polymerase chain reaction (rep‐PCR) fingerprinting and virulence gene amplification, suggesting that the population of E. ictaluri within catfish aquaculture in Mississippi and Alabama has been genetically stable over time. Plasmid profiling revealed the existence of at least four distinct plasmids among field isolates. Nevertheless, the E. ictaluri vaccine protected fish against all isolates, irrespective of heterogeneous or homogeneous plasmid profiles. In addition, plasmid profiles were supported by antimicrobial sensitivity testing, indicating that the E. ictaluri vaccine strain does not carry natural or plasmid‐mediated multidrug resistance to the three antibiotics approved for use in U.S. food fish aquaculture. Challenge data from this study argue against the need for multivalent E. ictaluri vaccines to account for presumed antigenic or genetic variation among wild‐type E. ictaluri isolates as the vaccine was effective against all archived isolates.
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