A large-scale, multi-year microbial community survey of a freshwater trout aquaculture facility.

Aquaculture is an important tool for solving the growing worldwide food demand, but infectious diseases of farmed animals represent a serious roadblock to continued industry growth. Therefore, it is essential to understand the microbial communities that reside within the built environments of aquaculture facilities to identify reservoirs of bacterial pathogens and potential correlations between commensal species and specific disease agents. Here, we present the results from 3 years of sampling a commercial rainbow trout aquaculture facility. We observed that the microbial communities residing on the abiotic surfaces within the hatchery were distinct from those residing on the surfaces at the facility's water source as well as the production raceways, despite similar communities in the water column at each location. Also, a subset of the water community seeds the biofilm communities. Lastly, we detected a common fish pathogen, Flavobacterium columnare, within the hatchery, including at the source water inlet. Importantly, the relative abundance of this pathogen was correlated with clinical disease. Our results characterized the microbial communities in an aquaculture facility, established that the hatchery environment contains a unique community composition and demonstrated that a specific fish pathogen resides within abiotic surface biofilms and is seeded from the natural water source.

Detection of Zoonotic Bacteria and Paragonimus kellicotti in Red Swamp Crayfish (Procambarus clarkii) and the Assessment of Traditional Crayfish Boils.

ABSTRACT: Studies of red swamp crayfish (Procambarus clarkii) outside of the United States confirm the presence of a variety of zoonotic pathogens, but it is unknown whether these same pathogens occur in P. clarkii in the United States. The U.S. commercial crayfish industry generates $200 million yearly, underscoring the need to evaluate this consumer commodity. The study objectives were to evaluate specific zoonotic pathogens present on P. clarkii from Alabama and Louisiana, states in the southeastern United States, and to determine the effectiveness of traditional food preparation methods to reduce pathogens. Experiment A evaluated the presence of Escherichia coli, Salmonella, Staphylococcus aureus, and Vibrio spp. in crayfish and environmental samples over a 2-month collection period (May to June 2021). Crayfish sampling consisted of swabbing the cephalothorax region; 15 samples were tested for E. coli, Salmonella, and S. aureus, and an additional 15 samples for Vibrio spp. Additionally, crayfish shipping materials were sampled. In experiment B, 92 crayfish were evaluated for Paragonimus kellicotti. Experiment C compared live and boiled crayfish for the presence of Vibrio spp. In experiments A and B, all 60 (100%) crayfish samples and 13 (81.25%) of 16 environmental samples showed growth characteristic of Vibrio spp. Three (5%) of 60 samples showed E. coli growth, with no statistical difference (P = 0.5536) between farms. P. kellicotti, Salmonella, and S. aureus were not recovered from any samples. In experiment C, all 10 (100%) of the live preboiled crayfish samples showed characteristic growth, whereas 1 (10%) of 10 samples of crayfish boiled in unseasoned water showed Vibrio growth (P < 0.0001). These results confirm that Vibrio spp. and E. coli may be present on U.S. commercial crayfish and that care should be taken when handling any materials that come into contact with live crayfish because they can potentially be contaminated.

Detecting Flavobacterial Fish Pathogens in the Environment via High-Throughput Community Analysis.

Diseases caused by the fish pathogens Flavobacterium columnare and Flavobacterium psychrophilum are major contributors of preventable losses in the aquaculture industry. The persistent and difficult-to-control infections caused by these bacteria make timely intervention and prophylactic elimination of pathogen reservoirs important measures to combat these disease-causing agents. In this study, we present two independent assays for detecting these pathogens in a range of environmental samples. Natural water samples were inoculated with F. columnare and F. psychrophilum over 5 orders of magnitude, and pathogen levels were detected using Illumina MiSeq sequencing and droplet digital PCR. Both detection methods accurately identified pathogen-positive samples and showed good agreement in quantifying each pathogen. Additionally, the real-world application of these approaches was demonstrated using environmental samples collected at a rainbow trout (Oncorhynchus mykiss) aquaculture facility. These results show that both methods can serve as useful tools for surveillance efforts in aquaculture facilities, where the early detection of these flavobacterial pathogens may direct preventative measures to reduce disease occurrence. IMPORTANCE Early detection of a deadly disease outbreak in a population can be the difference between mass mortality or mitigated effects. In the present study, we evaluated and compared two molecular techniques for detecting economically impactful aquaculture pathogens. We demonstrate that one of these techniques, 16S rRNA gene sequencing using Illumina MiSeq technology, provides the ability to accurately detect two freshwater fish pathogens, F. columnare and F. psychrophilum, while simultaneously profiling the native microbial community. The second technique, droplet digital PCR, is commonly used for pathogen detection, and the results obtained using the assays we designed with this method served to validate those obtained using the MiSeq method. These two methods offer distinct advantages. The MiSeq method pairs pathogen detection and microbial community profiling to answer immediate and long-term fish health concerns, while the droplet digital PCR method provides fast and highly sensitive detection that is useful for surveillance and rapid clinical responses.

Draft Genome Sequence of Janthinobacterium lividum ID1246, Isolated from a Rainbow Trout Hatchery Biofilm.

We present a draft genome sequence of Janthinobacterium lividum strain ID1246, isolated from within a rainbow trout hatchery raceway. Janthinobacterium spp. are well-known producers of antimicrobial compounds. Due to the unique isolation source, this genome may yield novel biosynthetic gene clusters.

Draft Genome Sequence of Aeromonas popoffii ID682, Isolated from a Natural Water Source in Idaho.

A draft genome sequence of Aeromonas popoffii ID682, isolated from a natural water source in Idaho, is presented here. A. popoffii is a relatively understudied species within a diverse, expanding freshwater genus of bacteria. Here, we describe only the second genome published for this species to date.

Arrested Development of Henneguya ictaluri (Cnidaria: Myxobolidae) in ♀ Channel Catfish × â™‚ Blue Catfish Hybrids.

Henneguya ictaluri is the etiologic agent of proliferative gill disease (PGD) in farm-raised Channel Catfish Ictalurus punctatus and hybrid catfish in the southeastern United States, and significant annual losses are attributed to this disease. Research suggests that H. ictaluri infection dynamics in Blue Catfish I. furcatus and hybrid catfish (Channel Catfish × Blue Catfish) differ from those in Channel Catfish. Two separate infectivity trials were conducted to investigate H. ictaluri development in Channel Catfish, Blue Catfish, and their hybrids. On two separate occasions with two different year-classes, fish were exposed to pond water containing H. ictaluri actinospores and sampled weekly for 12 weeks (trial 1) or 14 weeks (trial 2). In trial 1, the presence of H. ictaluri was evaluated histologically and by quantitative PCR of fish tissues, including gills, blood, anterior kidney, brain, heart, liver, posterior kidney, spleen, and stomach. Henneguya ictaluri DNA was detected in significantly higher concentrations throughout multiple organ systems in the Channel Catfish compared to the hybrid catfish and Blue Catfish, with the gills having higher quantities. Myxospores were observed in Channel Catfish gill tissue at 8 weeks postexposure. No myxospores were observed in Blue Catfish or hybrid catfish. The second trial focused on gills only and yielded similar results, with Channel Catfish having significantly greater H. ictaluri DNA quantities than hybrids or Blue Catfish across all time points. Myxospores were observed in Channel Catfish beginning at 6 weeks postexposure and were found in 36% (58/162) of Channel Catfish sampled for molecular and histological analysis during weeks 6-14. Myxospores in hybrid catfish were sparse, with single pseudocysts observed in two hybrid catfish (1.2%) at 14 weeks postexposure. These results imply arrested development of H. ictaluri in hybrid catfish. As such, culture of hybrid catfish may be an effective management strategy to minimize the burden of PGD.

Francisella marina sp. nov., Etiologic Agent of Systemic Disease in Cultured Spotted Rose Snapper (Lutjanus guttatus) in Central America.

Historically, piscine francisellosis in various warm-, temperate-, and cold-water fish hosts has been attributed to Francisella noatunensis From 2015 to 2016, an undescribed Francisella sp. was recovered during mortality events in cultured spotted rose snapper (Lutjanus guttatus) off the Pacific coast of Central America. Despite high mortality and emaciation, limited gross findings were observed in affected fish. Histological examination revealed multifocal granulomatous lesions, with the presence of numerous small, pleomorphic coccobacilli, predominantly in the peritoneum, spleen, kidneys, liver, pancreas, heart, and intestine. Sequencing of an ∼1,400-bp fragment of the 16S rRNA gene demonstrated these isolates to be most similar (99.9% identity) to Francisella sp. isolate TX077308 cultured from seawater in the Gulf of Mexico, while sharing <99% similarity to other Fransicella spp. Biochemical analysis, multilocus sequence comparisons of select housekeeping genes, repetitive extragenic palindromic PCR fingerprinting, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and fatty acid methyl ester analysis revealed marked differences between these isolates and other described members of the genus. Koch's postulates were fulfilled by experimental intracoelomic injection and immersion trials using Nile (Oreochromis niloticus) and blue (Oreochromis aureus) tilapia. Based on observed phenotypic and genotypic differences from recognized Francisella spp., the name Francisellamarina sp. nov. (NRRL B-65518) is proposed to accommodate these novel strains.IMPORTANCE Finfish aquaculture is the fastest growing global food production sector. Infectious disease, particularly emergent pathogens, pose a significant threat to established and nascent aquaculture industries worldwide. Herein, we characterize a novel pathogen isolated from mortality events in cultured spotted rose snapper in Central America. The bacteria recovered from these outbreaks were genetically and phenotypically dissimilar from other known Francisella spp. from fish, representing a previously unrecognized member of the genus Francisella, for which the name Francisella marina sp. nov. is proposed.

HYPERMUCOVISCOUS KLEBSIELLA PNEUMONIAE ISOLATES FROM STRANDED AND WILD-CAUGHT MARINE MAMMALS OF THE US PACIFIC COAST: PREVALENCE, PHENOTYPE, AND GENOTYPE.

Emergent hypermucoviscous (HMV) strains of Klebsiella pneumoniae have been reported in multiple marine mammal species; however, there is limited information regarding the epidemiology and pathogenesis of this infection in these species. We determined the prevalence of HMV K. pneumoniae in wild-caught and stranded marine mammal populations on the US Pacific Coast. Samples were collected from 270 free-ranging California sea lions (CSLs; Zalophus californianus) captured at three discrete sampling sites and from 336 stranded marine mammals of various species. We recovered HMV K. pneumoniae only from CSLs, with a prevalence of 1.5% (4 of 275) in stranded animals, compared with 1.1% (3 of 270) in wild-caught animals. We assessed the phenotypic and genotypic variability of recovered HMV K. pneumoniae isolates recovered from CSLs ( n=11) and of archival HMV and non-HMV isolates from stranded marine mammals ( n=19). All but two HMV isolates were of the K2 serotype, whereas none of the non-HMV isolates belonged to this serotype. Of the HMV isolates, 96% (24 of 25) were PCR positive for the HMV-associated gene p- rmpA, whereas 92% (23 of 25) were PCR positive for p- rmpA2. Genetic fingerprinting by repetitive extragenic palindromic PCR showed four discrete clusters, demonstrating genotypic variability that loosely correlated with phenotype. Antimicrobial susceptibility testing revealed all isolates from stranded CSLs were susceptible to ceftiofur, indicating this antimicrobial agent is an appropriate choice for treatment of HMV K. pneumoniae infections in stranded CSLs. Our culture assay could reliably detect HMV K. pneumoniae from concentrations as low as 102 colony-forming units per milligram of feces. We identified the presence of HMV K. pneumoniae in both wild-caught and stranded CSLs from the US Pacific Coast and highlight the need for further studies to evaluate the potential impact of this pathogen on marine mammal health.

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.

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.

Complete Genome Sequence of Edwardsiella ictaluri Isolate RUSVM-1 Recovered from Nile Tilapia (Oreochromis niloticus) in the Western Hemisphere.

Edwardsiella ictaluri is a Gram-negative bacillus that has recently been implicated in disease outbreaks in tilapia and zebrafish. We report here the complete and annotated genome sequence of an isolate from a Nile tilapia (Oreochromis niloticus), which contains a chromosome of 3,630,639 bp and two plasmids.

Diversity of Veronaea botryosa from different hosts and evaluation of laboratory challenge models for phaeohyphomycosis in Acipenser transmontanus.

Veronaea botryosa has been identified as a pathogen of cultured white sturgeon Acipenser transmontanus. In 2015, samples from 19 white sturgeon were received for diagnosis, of which 14 cultured positive for V. botryosa. Intraspecific variability among V. botryosa isolates from different clinically affected hosts and geographic regions was investigated using repetitive extragenic palindromic PCR fingerprinting (rep-PCR). The rep-PCR profiles of 16 V. botryosa isolates from a human, sea turtles, and cultured fish were distinct from those of other phaeoid fungi belonging to the genera Cladophialophora and Exophiala. To gain a better understanding of the pathogenesis of V. botryosa mycosis, 5 laboratory challenge methods were evaluated in white sturgeon fingerlings. Intramuscular (IM) and intracoelomic (IC) injection challenges produced cumulative mortalities of 13.3% (8/60) and 3.3% (2/60), respectively, and V. botryosa was recovered from 100% (10/10) of dead fingerlings. Affected fish exhibited abnormal orientation and/or failure to maintain neutral buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerated skin. After 6 wk, surviving fish were euthanized, and samples of liver were taken for mycological evaluation. Viable fungus was detected in 90% and 100% of fish surviving IM and IC challenge, respectively. No V. botryosa-associated mortality was detected in other groups challenged by immersion, immersion with abrasion, or orally. Both IM and IC challenge routes appear suitable for the induction of V. botryosa infection in white sturgeon and can serve as models for the study of disease pathogenesis associated with this emergent pathogen.

Complete Genome Sequence of Edwardsiella hoshinae ATCC 35051.

Edwardsiella hoshinae is a Gram-negative facultative anaerobe that has primarily been isolated from avians and reptiles. We report here the complete and annotated genome sequence of an isolate from a monitor lizard (Varanus sp.), which contains a chromosome of 3,811,650 bp and no plasmids.

Complete Genome Sequence of Edwardsiella piscicida Isolate S11-285 Recovered from Channel Catfish (Ictalurus punctatus) in Mississippi, USA.

Edwardsiella piscicida is a recently described Gram-negative facultative anaerobe and an important pathogen to many wild and cultured fish species worldwide. Here, we report the complete and annotated genome of E. piscicida isolate S11-285 recovered from channel catfish (Ictalurus punctatus), consisting of a chromosome of 3,923,603 bp and 1 plasmid.

Austrodiplostomum sp., Bolbophorus sp. (Digenea: Diplostomidae), and Clinostomum marginatum (Digenea: Clinostomidae) metacercariae in inland silverside Menidia beryllina from catfish aquaculture ponds, with notes on the infectivity of Austrodiplostomum sp. cercariae in channel catfish Ictalurus punctatus.

In the southeastern USA, catfish aquaculture is burdened by predation from piscivorous birds and the digenetic trematodes they carry. In addition to cultured ictalurid fish, other forage or incidental fish species inhabit catfish production ponds. Of these, the inland silverside Menidia beryllina was recently found to harbor larval metacercariae of several trematode species. Three species of metacercariae were reported, two of which represent the first morphological descriptions of an Austrodiplostomum sp. and Bolbophorus sp. metacercaria, respectively. A total of 15 silversides were collected from a commercial catfish pond and examined for trematode infection. These fish were parasitized by metacercariae of an Austrodiplostomum sp. (100 % prevalence) in the eyes and brain, a Bolbophorus sp. (86.7 % prevalence) in the musculature, and Clinostomum marginatum (33.3 % prevalence) in the musculature and fins. All three trematode species were characterized morphologically and molecularly by sequencing of the cytochrome c oxidase subunit 1 gene (CO1). In addition, the internal transcribed spacer 1 region (ITS1), 5.8S rRNA gene, and ITS2 region were determined for the Bolbophorus sp., which linked this metacercaria to a Bolbophorus sp. cercaria from a planorbid snail Planorbella trivolvis and an unnamed Bolbophorus sp. adult from the American white pelican Pelecanus erythrorhynchos. Furthermore, Biomphalaria havanensis snails were collected from the same pond and found actively shedding cercariae morphologically and molecularly consistent with a diplostomid cercaria reported from Bi . havanensis in catfish ponds in Mississippi, USA. Sequence comparisons deemed these cercariae conspecific to the Austrodiplostomum sp. from inland silverside described here. Channel catfish fingerlings were exposed to these cercariae at doses of 50 and 100 cercariae per fish. The infectivity of this Austrodiplostomum sp. in channel catfish was assessed at 10 and 20 days post exposure (dpe). Metacercariae were observed in both the eyes and brain of infected channel catfish, supporting molecular data that suggests the cercaria and metacercaria are both stages of a previously unidentified Austrodiplostomum sp. life cycle.

Histologic and molecular characterization of Edwardsiella piscicida infection in largemouth bass (Micropterus salmoides).

The genus Edwardsiella is composed of a diverse group of facultative anaerobic, gram-negative bacteria that can produce disease in a wide variety of hosts, including birds, reptiles, mammals, and fish. Our report describes the isolation and identification of Edwardsiella piscicida associated with chronic mortality events in 2 separate captive largemouth bass (Micropterus salmoides) populations in New York and Florida. Wet-mount biopsies of skin mucus, gill, kidney, and spleen from several affected largemouth bass contained significant numbers of motile bacteria. Histologic examination revealed multifocal areas of necrosis scattered throughout the heart, liver, anterior kidney, posterior kidney, and spleen. Many of the necrotic foci were encapsulated or replaced by discrete granulomas and associated with colonies of gram-negative bacteria. Initial phenotypic and matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis against existing spectral databases of recovered isolates identified these bacteria as Edwardsiella tarda Subsequent molecular analysis using repetitive sequence mediated and species-specific PCR, as well as 16S rRNA, rpoB, and gyrB sequences, classified these isolates as E. piscicida As a newly designated taxon, E. piscicida should be considered as a differential for multiorgan necrosis and granulomas in largemouth bass.

Complete Genome Sequence of an Edwardsiella piscicida-Like Species, Recovered from Tilapia in the United States.

An Edwardsiella piscicida-like species is a Gram-negative facultative anaerobe that causes disease in some fish species. In this report, we present the complete and annotated genome of isolate LADL05-105, recovered from cultured tilapia reared in Louisiana, which contains a chromosome of 4,142,037 bp and no plasmids.

Complete Genome Sequence of an Edwardsiella piscicida-Like Species Isolated from Diseased Grouper in Israel.

The Edwardsiella piscicida-like sp. is a Gram-negative facultative anaerobe that causes disease in some fish species. We report here the complete genome sequence of a virulent isolate from a diseased white grouper (Epinephelus aeneus) raised on the Red Sea in Israel, which contains a chromosome of 3,934,167 bp and no plasmids.

Complete Genome Sequence of Edwardsiella tarda Isolate FL95-01, Recovered from Channel Catfish.

Edwardsiella tarda is a Gram-negative facultative anaerobe that has been isolated from fish, reptiles, amphibians, and mammals, including humans. This is a report of the complete and annotated genome of isolate FL95-01, recovered from channel catfish (Ictalurus punctatus).

Real-time polymerase chain reaction assays for the detection and quantification of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella piscicida-like species in catfish tissues and pond water.

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.

Comparative susceptibility of Channel Catfish, Blue Catfish, and their hybrid cross to experimental challenge with Bolbophorus damnificus (Digenea: Bolbophoridae) cercariae.

The digenetic trematode Bolbophorus damnificus has been implicated in significant losses in catfish aquaculture since the late 1990s. The complex life cycle sequentially involves the American white pelican Pelecanus erythrorhynchos, the marsh rams horn snail Planorbella trivolvis, and Channel Catfish Ictalurus punctatus. Research supports anecdotal reports from the industry, suggesting that the hybrid of Channel Catfish×Blue Catfish I. furcatus is less susceptible to disease agents that have been historically prohibitive to Channel Catfish production, namely the gram-negative bacteria Edwardsiella ictaluri and Flavobacterium columnare, as well as the myxozoan parasite Henneguya ictaluri. This current research compared the susceptibility of Channel Catfish, Blue Catfish, and their hybrid cross to an experimental challenge by B. damnificus. Fish were exposed to 0, 100, 200, and 400 B. damnificus cercariae per fish, and the numbers of metacercariae per fish were determined 14 d postchallenge. Metacercariae were recovered from all challenged fish. There were no significant differences among fish groups challenged with the same dose, suggesting Channel and Blue Catfish and their hybrid are comparably susceptible to B. damnificus infection. As such, it is recommended that producers raising hybrid catfish remain diligent in controlling populations of the snail intermediate host to prevent production losses attributed to B. damnificus, especially when loafing pelicans have been observed at the aquaculture operation.