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.

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The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm-raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive-sequence-mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep-PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep-PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.

Members of the genus Edwardsiella are important pathogens of cultured and wild fish globally. Recent investigations into the phenotypic and genotypic variation of Edwardsiella tarda have led to the segregation of E. tarda into three distinct taxa: E. tarda, Edwardsiella piscicida, and Edwardsiella anguillarum. In catfish aquaculture in the southeastern USA, E. piscicida has been more commonly associated with disease than E. tarda or E. anguillarum, and recent research has demonstrated E. piscicida to be more pathogenic in channel catfish than E. tarda or E. anguillarum. Anecdotal reports from industry suggest an increased prevalence of E. piscicida associated with the culture of channel (♀) × blue (♂) hybrid catfish. This work investigated the comparative susceptibility of channel catfish, blue catfish, and their hybrid cross to molecularly confirmed isolates of E. tarda, E. piscicida, and E. anguillarum. There was significantly higher mortality in hybrid catfish compared to channel catfish following intracoelomic injection of E. piscicida. To our knowledge, E. piscicida is the first bacterial pathogen to demonstrate increased pathogenicity in hybrid catfish compared to channel catfish. KEYWORDS blue catfish, channel catfish, Edwardsiella, hybrid catfish

There is a trend toward the increased incidence and prevalence of Edwardsiella piscicida septicemia in US catfish aquaculture, particularly in channel ♀, Ictalurus punctatus, × blue ♂, I. furcatus, hybrid catfish. From 2013 to 2017, a total of 3242 disease case submissions were made to the Aquatic Research and Diagnostic Laboratory (ARDL) at the Thad Cochran National Warmwater Aquaculture Center in Stoneville, MS. Of these, 1400 (43.2%) were hybrids. E. piscicida was suspected in 138 (4.3%) of cases, the majority of which (89.1%) were from hybrid catfish. A molecular survey of these isolates confirmed the majority (92.0%) to be E. piscicida. Furthermore, cases of E. piscicida from hybrids submitted to the ARDL and the Aquatic Diagnostic Laboratory of the Mississippi State University College of Veterinary Medicine in Starkville, MS, were documented for gross lesions and histological analysis. Grossly, E. piscicida presents with small dermal ulcerations, a raised fluid‐filled cranial midline lesion that is frequently ulcerated, hemorrhage in the gills, exophthalmia, and abdominal distension. Internally, lesions include splenomegaly, straw‐colored ascites, renomegaly, and occasionally hemorrhagic intestines. Histopathological examination is in agreement with gross observations, and infected fish repeatedly demonstrate a mononuclear meningoencephalitis, hemorrhagic branchitis, splenitis, ulcerative dermatitis, granulomatous interstitial nephritis, and hepatitis coupled with a hemorrhagic enteritis.

Since 2012, low-to-moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram-positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep-PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.

Abstract.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 10 3 (E. piscicida) to 10 2 (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. 13 Speciesspecific polymerase chain reaction (PCR) assays targeting the fimbrial gene cluster were developed for each individual taxa and were demonstrated specific to their respective target organisms. 13,26 In the current study, real-time PCR (qPCR) assays were developed using these established primer sets and were validated for the detection and quantification of E. tarda, E. piscicida, and E. piscicida-like sp. from catfish kidney tissues, pond water, and broth culture. Materials and methods Bacterial cultures and isolation of genomic DNAThe Edwardsiella strains used in the validation of the assays in the current study were characterized as part of an earlier study 13 and identified by gyrB sequencing and speciesspecific PCR (Table 1). In addition, an Edwardsiella hoshinae strain (ATCC 35051), an Escherichia coli strain (ATCC 25922), 2 Flavobacterium columnare strains (94-081 and ATCC 49512), and 2 Aeromonas hydrophila strains (ML 09-119 and TN 97-08), including a highly virulent strain (ML 09-119) attributed to disease outbreaks in farm-raised catfish 18 were also included in the validation process. Design of primer and probe setsThe development of the qPCR assays specific to E. tarda, E. piscicida, and E. piscicida-like sp. was based on previously published PCR primers. 13,26 Oligonucleotide probes corresponding to each primer set were designed using primer design software 25 and synthesized...

A mortality event in cultured white sturgeon Acipenser transmontanus (Richardson, 1836) sub-adults was investigated. After transfer between farms, high mortality was observed in fish, associated with back arching, abnormal swimming, and ulcerative skin lesions. Necropsy of moribund individuals revealed hemorrhagic ascites and petechial hemorrhages in the coelomic peritoneum and serosa of internal organs. Acipenserid herpesvirus 2 (AciHV-2) was isolated from external tissue samples, then identified and genotyped by sequencing of the terminase and polymerase genes. In addition, Streptococcus iniae was recovered from internal organs of affected fish. Histologic changes were limited to interstitial hematopoietic areas of the kidney and consisted of small foci of necrosis accompanied by fibrin deposition, minimal inflammatory response, and small numbers of bacterial cocci compatible with streptococci. Identity was confirmed by partial sequencing of the 16S rRNA, rpoB, and gyrB genes. Genetic fingerprinting demonstrated a genetic profile distinct from S. iniae isolates recovered from previous outbreaks in wild and cultured fish in North America, South America, and the Caribbean. Although the isolates were resistant to white sturgeon complement in serum killing assays, in vivo challenges failed to fulfill Koch's postulates. However, the clinical presentation, coupled with consistent recovery of S. iniae and AciHV-2 from moribund fish, suggests viral and bacterial co-infection were the proximate cause of death. To our knowledge, this represents the first report of AciHV-2 and S. iniae co-infection in cultured white sturgeon.