Massive branchial henneguyosis of catfish: A distinct, myxozoan-induced gill disease caused by severe interlamellar Henneguya exilis infection in catfish aquaculture.

Proliferative gill disease (PGD), caused by the myxozoan Henneguya ictaluri, has been the most notorious parasitic gill disease in the US catfish aquaculture industry. In 2019, an unusual gill disease caused by massive burdens of another myxozoan, Henneguya exilis, was described in channel (Ictalurus punctatus) × blue (Ictalurus furcatus) hybrid catfish. Targeted metagenomic sequencing and in situ hybridization (ISH) were used to differentiate these conditions by comparing myxozoan communities involved in lesion development and disease pathogenesis between massive H. exilis infections and PGD cases. Thirty ethanol-fixed gill holobranchs from 7 cases of massive H. exilis infection in hybrid catfish were subjected to targeted amplicon sequencing of the 18S rRNA gene and compared to a targeted metagenomic data set previously generated from clinical PGD case submissions. Furthermore, serial sections of 14 formalin-fixed gill holobranchs (2 per case) were analyzed by RNAscope duplex chromogenic ISH assays targeting 8 different myxozoan species. Targeted metagenomic and ISH data were concordant, indicating myxozoan community compositions significantly differ between PGD and massive branchial henneguyosis. Although PGD cases often consist of mixed species infections, massive branchial henneguyosis consisted of nearly pure H. exilis infections. Still, H. ictaluri was identified by ISH in association with infrequent PGD lesions, suggesting coinfections occur, and some cases of massive branchial henneguyosis may contain concurrent PGD lesions contributing to morbidity. These findings establish a case definition for a putative emerging, myxozoan-induced gill disease of farm-raised catfish with a proposed condition name of massive branchial henneguyosis of catfish (MBHC).

Hypothalamic Transcriptome Response To Simulated Diel Earthen Pond Hypoxia Cycles In Channel Catfish (Ictalurus punctatus).

Commercial culture of channel catfish (Ictalurus punctatus) occurs in earthen ponds that are characterized by diel swings in dissolved oxygen concentration that can fall to severe levels of hypoxia which can suppress appetite and lead to suboptimal growth. Given the significance of the hypothalamus in regulating these processes in other fishes, an investigation into the hypothalamus transcriptome was conducted to identify specific genes and expression patterns responding to hypoxia. Channel catfish in normoxic water were compared to catfish subjected to 12 hours of hypoxia (20% oxygen saturation; 1.8 mg O2/L; 27 °C) followed by 12 hours of recovery in normoxia to mimic 24-hours in a catfish aquaculture pond. Fish were sampled at 0-, 6-, 12-, 18-, and 24-hour time points, with the 6- and 12-hour samplings occurring during hypoxia. A total of 190 genes were differentially expressed during the experiment, with most occurring during hypoxia and returning to baseline values within 6 hours of normoxia. Differentially expressed genes were sorted by function into Gene Ontology biological processes and revealed that most were categorized as "response to hypoxia", "sprouting angiogenesis", and "cellular response to xenobiotic stimulus". The patterns of gene expression reported here suggest that transcriptome responses to hypoxia are broad and quickly reversibly with the onset of normoxia. Although no genes commonly reported to modulate appetite were found to be differentially expressed in this experiment, several candidates were identified for future studies investigating the interplay between hypoxia and appetite in channel catfish, including adm, igfbp1a, igfbp7, and stc2b.

Morphological and molecular data establishes Clinostomum dolichorchum n. sp. (Digenea: Clinostomidae) in the great blue heron Ardea herodias L. and American bullfrog Rana catesbeiana Shaw.

Clinostomum is a cosmopolitan genus of trematodes that infect piscivorous birds, freshwater molluscs, freshwater fish and amphibians. Herein, a novel species of Clinostomum is described based on morphological and molecular data from an adult in the oral cavity of the great blue heron Ardea herodias and metacercariae collected from the gills and skin of American bullfrog tadpoles Rana catesbeiana. The novel species shares similar qualitative and quantitative morphological features with a congener, Clinostomum marginatum, which has overlap in host and geographic distribution. The most notable morphological difference when compared to C. marginatum is the greater posterior testis length of the novel species. Molecular data resolved similarities with morphological comparisons to nominal species and supports the establishment of a novel species. Molecular data include partial small ribosomal subunit (18S rRNA gene), ribosomal internal transcribed spacer regions (ITS1, 5.8S rRNA gene, and ITS2), partial large ribosomal subunit (28S rRNA gene), cytochrome c oxidase subunit 1 gene (cox1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene (nad1) sequences. Phylogenetic analyses place the novel species in a sister clade to C. marginatum. Morphological and molecular data, combined with phylogenetic analyses support the establishment of Clinostomum dolichorchum n. sp.

Redefining piscine lactococcosis.

Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae. However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)-L. garvieae, L. petauri, and L. formosensis-which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis. Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention. IMPORTANCE: Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

Complete genome of Vibrio harveyi isolate K2014767 from the hepatopancreas of captive Caribbean spiny lobster (Panulirus argus).

The complete genome sequence is reported for Vibrio harveyi isolate K2014767, isolated from a captive Caribbean spiny lobster (Panulirus argus) during a species-specific mortality event in a public display aquarium in the United States.

Acipenserid herpesvirus 2 genome and partial validation of a qPCR for its detection in white sturgeon Acipenser transmontanus.

White sturgeon Acipenser transmontanus is the primary species used for caviar and sturgeon meat production in the USA. An important pathogen of white sturgeon is acipenserid herpesvirus 2 (AciHV-2). In this study, 4 archived isolates from temporally discrete natural outbreaks spanning the past 30 yr were sequenced via Illumina and Oxford Nanopore Technologies platforms. Assemblies of approximately 134 kb were obtained for each isolate, and the putative ATPase subunit of the terminase gene was selected as a potential quantitative PCR (qPCR) target based on sequence conservation among AciHV-2 isolates and low sequence homology with other important viral pathogens. The qPCR was repeatable and reproducible, with a linear dynamic range covering 5 orders of magnitude, an efficiency of approximately 96%, an R2 of 0.9872, and an analytical sensitivity of 103 copies per reaction after 35 cycles. There was no cross-reaction with other known viruses or closely related sturgeon species, and no inhibition by sturgeon DNA. Clinical accuracy was assessed from white sturgeon juveniles exposed to AciHV-2 by immersion. Viral culture (gold standard) and qPCR were in complete agreement for both cell culture negative and cell culture positive samples, indicating that this assay has 100% relative accuracy compared to cell culture during an active outbreak. The availability of a whole-genome sequence for AciHV-2 and a highly specific and sensitive qPCR assay for detection of AciHV-2 in white sturgeon lays a foundation for further studies on host-pathogen interactions while providing a specific and rapid test for AciHV-2 in captive and wild populations.

Coinfection of channel catfish (Ictalurus punctatus) with virulent Aeromonas hydrophila and Flavobacterium covae exacerbates mortality.

Flavobacterium covae and virulent Aeromonas hydrophila are prevalent bacterial pathogens within the US catfish industry that can cause high mortality in production ponds. An assessment of in vivo bacterial coinfection with virulent A. hydrophila (ML09-119) and F. covae (ALG-00-530) was conducted in juvenile channel catfish (Ictalurus punctatus). Catfish were divided into seven treatments: (1) mock control; (2) and (3) high and low doses of virulent A. hydrophila; (4) and (5) high and low doses of F. covae; (6) and (7) simultaneous challenge with high and low doses of virulent A. hydrophila and F. covae. In addition to the mortality assessment, anterior kidney and spleen were collected to evaluate immune gene expression, as well as quantify bacterial load by qPCR. At 96 h post-challenge (hpc), the high dose of virulent A. hydrophila infection (immersed in 2.3 × 107 CFU mL-1 ) resulted in cumulative percent mortality (CPM) of 28.3 ± 9.5%, while the high dose of F. covae (immersed in 5.2 × 106 CFU mL-1 ) yielded CPM of 23.3 ± 12.9%. When these pathogens were delivered in combination, CPM significantly increased for both the high- (98.3 ± 1.36%) and low-dose combinations (76.7 ± 17.05%) (p < .001). Lysozyme activity was found to be different at 24 and 48 hpc, with the high-dose vAh group demonstrating greater levels than unexposed control fish at each time point. Three proinflammatory cytokines (tnfα, il8, il1b) demonstrated increased expression levels at 48 hpc. These results demonstrate the additive effects on mortality when these two pathogens are combined. The synthesis of these mortality and health metrics advances our understanding of coinfections of these two important catfish pathogens and will aid fish health diagnosticians and channel catfish producers in developing therapeutants and prevention methods to control bacterial coinfections.

Flavobacterium covae is the predominant species of columnaris-causing bacteria impacting the Channel Catfish industry in the southeastern United States.

OBJECTIVE: Columnaris disease is a leading cause of disease-related losses in the catfish industry of the southeastern United States. The term "columnaris-causing bacteria" (CCB) has been coined in reference to the four described species that cause columnaris disease: Flavobacterium columnare, F. covae, F. davisii, and F. oreochromis. Historically, F. columnare, F. covae, and F. davisii have been isolated from columnaris disease cases in the catfish industry; however, there is a lack of knowledge of which CCB species are most prevalent in farm-raised catfish. The current research objectives were to (1) sample columnaris disease cases from the U.S. catfish industry and identify the species of CCB involved and (2) determine the virulence of the four CCB species in Channel Catfish Ictalurus punctatus in controlled laboratory challenges. METHODS: Bacterial isolates or swabs of external lesions from catfish were collected from 259 columnaris disease cases in Mississippi and Alabama during 2015-2019. The DNA extracted from the samples was analyzed using a CCB-specific multiplex polymerase chain reaction to identify the CCB present in each diagnostic case. Channel Catfish were challenged by immersion with isolates belonging to each CCB species to determine virulence at ~28°C and 20°C. RESULT: Flavobacterium covae was identified as the predominant CCB species impacting the U.S. catfish industry, as it was present in 94.2% (n = 244) of diagnostic case submissions. Challenge experiments demonstrated that F. covae and F. oreochromis were highly virulent to Channel Catfish, with most isolates resulting in near 100% mortality. In contrast, F. columnare and F. davisii were less virulent, with most isolates resulting in less than 40% mortality. CONCLUSION: Collectively, these results demonstrate that F. covae is the predominant CCB in the U.S. catfish industry, and research aimed at developing new control and prevention strategies should target this bacterial species. The methods described herein can be used to continue monitoring the prevalence of CCB in the catfish industry and can be easily applied to other industries to identify which Flavobacterium species have the greatest impact.

Iron supplementation in the diets of hybrid catfish (Ictalurus punctatus × I. furcatus) juveniles affected haematocrit levels and potentially decreased disease resistance to Edwardsiella ictaluri.

To prevent catfish idiopathic anaemia, diets fortified with iron have been adopted as a regular practice on commercial catfish farms to promote erythropoiesis. However, the effects of prolonged exposure of excess dietary iron on production performance and disease resistance for hybrid catfish (Ictalurus punctatus × I. furcatus) remains unknown. Four experimental diets were supplemented with ferrous monosulphate to provide 0, 500, 1000, and 1500 mg of iron per kg of diet. Groups of 16 hybrid catfish juveniles (~22.4 g) were stocked in each of 20, 110-L aquaria (n = 5), and experimental diets were offered to the fish to apparent satiation for 12 weeks. At the end of the study, production performance, survival, condition indices, as well as protein and iron retention were unaffected by the dietary treatments. Blood haematocrit and the iron concentration in the whole-body presented a linear increase with the increasing the dietary iron. The remaining fish from the feeding trial was challenged with Edwardsiella ictaluri. Mortality was mainly observed for the dietary groups treated with iron supplemented diets. The results for this study suggest that iron supplementation beyond the required levels does affect the blood production, and it may increase their susceptibility to E. ictaluri infection.

Influence of probiotic and prebiotic supplementation on intestinal microbiota and resistance to Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus) following florfenicol administration.

Enteric septicemia of catfish (ESC), caused by the gram-negative enteric bacteria Edwardsiella ictaluri, is a significant threat to catfish aquaculture in the southeastern United States. Antibiotic intervention can reduce mortality; however, antibiotic use results in an imbalance, or dysbiosis, of the gut microbiota, which may increase susceptibility of otherwise healthy fish to enteric infections. Herein, recovery of the intestinal microbiota and survivability of channel catfish in response to ESC challenge was evaluated following a 10-day course of florfenicol and subsequent probiotic or prebiotic supplementation. Following completion of florfenicol therapy, fish were transitioned to a basal diet or diets supplemented with a probiotic or prebiotic for the remainder of the study. Digesta was collected on Days 0, 4, 8 and 12, beginning on the first day after cessation of antibiotic treatment, and gut microbiota was characterized by Illumina sequencing of the 16S rRNA gene (V4 region). Remaining fish were challenged with E. ictaluri and monitored for 32 days post-challenge. Florfenicol administration resulted in dysbiosis characterized by inflated microbial diversity, which began to recover in terms of diversity and composition 4 days after cessation of florfenicol administration. Fish fed the probiotic diet had higher survival in response to ESC challenge than the prebiotic (p = .019) and negative control (p = .029) groups.

Species-specific in situ hybridization confirms arrested development of Henneguya ictaluri in hybrid catfish (Channel Catfish × Blue Catfish) under experimental conditions, with notes on mixed-species infections in clinical cases of proliferative gill disease from Mississippi catfish aquaculture.

OBJECTIVE: Proliferative gill disease (PGD) in Channel Catfish Ictalurus punctatus and hybrid catfish (Channel Catfish × Blue Catfish I. furcatus) is attributed to the myxozoan Henneguya ictaluri. Despite evidence of decreased H. ictaluri transmission and impaired parasite development in hybrid catfish, PGD still occurs in hybrid production systems. Previous metagenomic assessments of clinical PGD cases revealed numerous myxozoans within affected gill tissues in addition to H. ictaluri. The objective of this study was to investigate the development and pathologic contributions of H. ictaluri and other myxozoans in naturally and experimentally induced PGD. METHODS: Henneguya species-specific in situ hybridization (ISH) assays were developed using RNAscope technology. Natural infections were sourced from diagnostic case submissions in 2019. Experimental challenges involved Channel Catfish and hybrid catfish exposed to pond water from an active PGD outbreak, and the fish were sampled at 1, 7, 10, 12, 14, 16, 18, and 20 weeks postchallenge. RESULT: Nine unique ISH probes were designed, targeting a diagnostic variable region of the 18S ribosomal RNA gene of select myxozoan taxa identified in clinical PGD cases. Partial validation from pure H. ictaluri, H. adiposa, H. postexilis, and H. exilis infections illustrated species-specific labeling and no cross-reactivity between different myxozoan species or the catfish hosts. After experimental challenge, mature plasmodia of H. ictaluri and H. postexilis formed in Channel Catfish but were not observed in hybrids, suggesting impaired or delayed sporogenesis in the hybridized host. These investigations also confirmed the presence of mixed infections in clinical PGD cases. CONCLUSION: Although H. ictaluri appears to be the primary cause of PGD, presporogonic stages of other myxozoans were also present, which may contribute to disease pathology and exacerbate respiratory compromise by further altering normal gill morphology. This work provides molecular confirmation and more resolute developmental timelines of H. ictaluri and H. postexilis in Channel Catfish and supports previous research indicating impaired or precluded H. ictaluri sporogony in hybrid catfish.

spaB-positive Erysipelothrix rhusiopathiae, a novel teleost pathogen isolated from cultured barramundi.

Members of the genus Erysipelothrix are emergent pathogens of cultured eels, as well as several characid and cyprinid species. Since 2013, E. rhusiopathiae has been reported from diseased barramundi (Lates calcarifer) cultured in North America; we recovered 8 E. rhusiopathiae isolates from diseased fish during different outbreaks from the same farm. The E. rhusiopathiae isolates from barramundi were compared phenotypically and genetically to E. piscisicarius isolates characterized from ornamental fish and E. rhusiopathiae recovered from aquatic and terrestrial animals. All barramundi isolates were PCR-positive for the surface protective antigen type B (spaB) gene, and shared ≥ 99.7% sequence similarity among concatenated multilocus sequence analysis gene sequences, indicating a high degree of genetic homogeneity. These isolates were > 99% similar to other spaB-positive isolates from marine invertebrates and marine mammals, consistent with findings for other spa types. The spaA and spaB isolates shared < 98% similarity, as well as < 90% similarity with spaC-positive E. piscisicarius. Similar clonality among the spaB isolates was observed using repetitive element palindromic PCR. In experimental intracoelomic injection challenges conducted to fulfill Koch postulates, 67% of exposed tiger barbs (Puntigrus tetrazona) died within 14 d of challenge. Our study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and the emergence of members of the genus Erysipelothrix as nascent fish pathogens.

Effect of hypoxia duration and pattern on channel Catfish (Ictalurus punctatus) neuropeptide gene expression and hematology.

Commercial aquaculture production of channel catfish (Ictalurus punctatus) occurs in shallow ponds with daily cycling of dissolved oxygen concentration ranging from supersaturation to severe hypoxia. Once daily minimum dissolved oxygen concentration falls below 3.0 mg O2/L, channel catfish have a reduced appetite, leading to reduced growth rates. In other fishes, upregulation of the neuropeptides corticotropin-releasing factor (CRF) and urotensin I (UI) have been implicated as initiating the mechanism responsible for decreasing appetite once an environmental stressor is detected. Channel catfish maintained at 27 °C in aquaria were subjected to varying durations and patterns of hypoxia (1.75 ± 0.07 mg O2/L) to evaluate underlying physiological responses to hypoxia and determine if hypothalamic CRF and UI are responsible for hypoxia-induced anorexia in channel catfish. During a short exposure to hypoxia (12 h), venous PO2 was significantly lower within 6 h and was coupled with an increase of hematocrit and decrease of blood osmolality, yet all responses reversed within 12 h after returning to normoxia. When this pattern of hypoxia and normoxia was repeated cyclically for 5 days, these physiological responses repeated daily. Extended periods of hypoxia (5 days) resulted in similar hematological responses, which did not recover to baseline values during the hypoxia exposure. This study did not find a significant change in hypothalamic transcription of CRF and UI during hypoxia challenges but did identify multiple physiological adaptive responses that work together to reduce the severity of experimentally induced hypoxia in channel catfish.

Edwardsiella tarda Isolated from a Kidney Mass in a Common Loon (Gavia immer).

A Common Loon (Gavia immer) was found recumbent at Island Beach State Park, New Jersey, US, and euthanized. Necropsy revealed a caseous mass in the kidney, from which bacteria were isolated and phenotypically and molecularly identified as Edwardsiella tarda.

Vibrio harveyi in a Caribbean spiny lobster (Panulirus argus) with hepatopancreas necrosis.

A Caribbean spiny lobster, Panulirus argus, was submitted for necropsy after a number of species-specific fatalities in a public aquarium. The hemolymph was opaque and did not clot and the hepatopancreas had multiple foci of necrosis centered on gram-negative bacteria. Pure cultures of Vibrio harveyi were isolated, identified initially by matrix laser desorption ionization-time of flight mass spectrometry, and confirmed by multilocus sequencing of the gyrB, recA, rpoA, and pyrH genes. As Caribbean spiny lobsters continue to be used for consumption and displayed in public aquariums, chronicling potential pathogens is warranted to inform differential diagnoses and to develop management strategies to reduce incidence of infectious disease in captive populations.

The Infection Dynamics of Experimental Edwardsiella ictaluri and Flavobacterium covae Coinfection in Channel Catfish (Ictalurus punctatus).

Edwardsiella ictaluri and Flavobacterium covae are pervasive bacterial pathogens associated with significant losses in catfish aquaculture. Bacterial coinfections have the potential to increase outbreak severity and can worsen on-farm mortality. A preliminary assessment of in vivo bacterial coinfection with E. ictaluri (S97-773) and F. covae (ALG-00-530) was conducted using juvenile channel catfish (Ictalurus punctatus). Catfish were divided into five treatment groups: (1) mock control; (2) E. ictaluri full dose (immersion; 5.4 × 105 CFU mL-1); (3) F. covae full dose (immersion; 3.6 × 106 CFU mL-1); (4) E. ictaluri half dose (immersion; 2.7 × 105 CFU mL-1) followed by half dose F. covae (immersion; 1.8 × 106 CFU mL-1); and (5) F. covae half dose followed by half dose E. ictaluri. In the coinfection challenges, the second inoculum was delivered 48 h after the initial exposure. At 21 days post-challenge (DPC), the single dose E. ictaluri infection yielded a cumulative percent mortality (CPM) of 90.0 ± 4.1%, compared with 13.3 ± 5.9% in the F. covae group. Mortality patterns in coinfection challenges mimicked the single dose E. ictaluri challenge, with CPM of 93.3 ± 5.4% for fish initially challenged with E. ictaluri followed by F. covae, and 93.3 ± 2.7% for fish exposed to F. covae and subsequently challenged with E. ictaluri. Despite similarities in the final CPM within the coinfection groups, the onset of peak mortality was delayed in fish exposed to F. covae first but was congruent with mortality trends in the E. ictaluri challenge. Catfish exposed to E. ictaluri in both the single and coinfected treatments displayed increased serum lysozyme activity at 4-DPC (p < 0.001). Three pro-inflammatory cytokines (il8, tnfα, il1ß) were evaluated for gene expression, revealing an increase in expression at 7-DPC in all E. ictaluri exposed treatments (p < 0.05). These data enhance our understanding of the dynamics of E. ictaluri and F. covae coinfections in US farm-raised catfish.

Pathology, microbiology, and genetic diversity associated with Erysipelothrix rhusiopathiae and novel Erysipelothrix spp. infections in southern sea otters (Enhydra lutris nereis).

Erysipelothrix spp., including E. rhusiopathiae, are zoonotic bacterial pathogens that can cause morbidity and mortality in mammals, fish, reptiles, birds, and humans. The southern sea otter (SSO; Enhydra lutris nereis) is a federally-listed threatened species for which infectious disease is a major cause of mortality. We estimated the frequency of detection of these opportunistic pathogens in dead SSOs, described pathology associated with Erysipelothrix infections in SSOs, characterized the genetic diversity and antimicrobial susceptibility of SSO isolates, and evaluated the virulence of two novel Erysipelothrix isolates from SSOs using an in vivo fish model. From 1998 to 2021 Erysipelothrix spp. were isolated from six of >500 necropsied SSOs. Erysipelothrix spp. were isolated in pure culture from three cases, while the other three were mixed cultures. Bacterial septicemia was a primary or contributing cause of death in five of the six cases. Other pathology observed included suppurative lymphadenopathy, fibrinosuppurative arteritis with thrombosis and infarction, bilateral uveitis and endophthalmitis, hypopyon, petechia and ecchymoses, mucosal infarction, and suppurative meningoencephalitis and ventriculitis. Short to long slender Gram-positive or Gram-variable bacterial rods were identified within lesions, alone or with other opportunistic bacteria. All six SSO isolates had the spaA genotype-four isolates clustered with spaA E. rhusiopathiae strains from various terrestrial and marine animal hosts. Two isolates did not cluster with any known Erysipelothrix spp.; whole genome sequencing revealed a novel Erysipelothrix species and a novel E. rhusiopathiae subspecies. We propose the names Erysipelothrix enhydrae sp. nov. and Erysipelothrix rhusiopathiae ohloneorum ssp. nov. respectively. The type strains are E. enhydrae UCD-4322-04 and E. rhusiopathiae ohloneorum UCD-4724-06, respectively. Experimental injection of tiger barbs (Puntigrus tetrazona) resulted in infection and mortality from the two novel Erysipelothrix spp. Antimicrobial susceptibility testing of Erysipelothrix isolates from SSOs shows similar susceptibility profiles to isolates from other terrestrial and aquatic animals. This is the first description of the pathology, microbial characteristics, and genetic diversity of Erysipelothrix isolates recovered from diseased SSOs. Methods presented here can facilitate case recognition, aid characterization of Erysipelothrix isolates, and illustrate assessment of virulence using fish models.

First Isolation of a Herpesvirus (Family Alloherpesviridae) from Great Lakes Lake Sturgeon (Acipenser fulvescens).

The lake sturgeon (Acipenser fulvescens; LST) is the only native sturgeon species in the Great Lakes (GL), but due to multiple factors, their current populations are estimated to be <1% of historical abundances. Little is known about infectious diseases affecting GL-LST in hatchery and wild settings. Therefore, a two-year disease surveillance study was undertaken, resulting in the detection and first in vitro isolation of a herpesvirus from grossly apparent cutaneous lesions in wild adult LST inhabiting two GL watersheds (Erie and Huron). Histological and ultrastructural examination of lesions revealed proliferative epidermitis associated with herpesvirus-like virions. A virus with identical ultrastructural characteristics was recovered from cells inoculated with lesion tissues. Partial DNA polymerase gene sequencing placed the virus within the Family Alloherpesviridae, with high similarity to a lake sturgeon herpesvirus (LSHV) from Wisconsin, USA. Genomic comparisons revealed ~84% Average Nucleotide Identity between the two isolates, leading to the proposed classification of LSHV-1 (Wisconsin) and LSHV-2 (Michigan) for the two viruses. When naïve juvenile LST were immersion-exposed to LSHV-2, severe disease and ~33% mortality occurred, with virus re-isolated from representative skin lesions, fulfilling Rivers' postulates. Results collectively show LSHV-2 is associated with epithelial changes in wild adult LST, disease and mortality in juvenile LST, and is a potential threat to GL-LST conservation.

Evaluation of Edwardsiella piscicida basS and basR mutants as vaccine candidates in catfish against edwardsiellosis.

Catfish farming is the largest aquaculture industry in the United States and an important economic driver in several southeastern states. Edwardsiella piscicida is a Gram-negative pathogen associated with significant losses in catfish aquaculture. Several Gram-negative bacteria use the BasS/BasR two-component system (TCS) to adapt to environmental changes and the host immune system. Currently, the role of BasS/BasR system in E. piscicida virulence has not been characterized. In the present study, two mutants were constructed by deleting the basS and basR genes in E. piscicida strain C07-087. Both mutant strains were characterized for virulence and immune protection in catfish hosts. The EpΔbasS and EpΔbasR mutants were more sensitive to acidic environments and produced significantly less biofilm than the wild-type. In vivo studies in channel catfish (Ictalurus punctatus) revealed that both EpΔbasS and EpΔbasR were significantly attenuated compared with the parental wild-type (3.57% and 4.17% vs. 49.16% mortalities). Moreover, there was significant protection, 95.2% and 92.3% relative percent survival (RPS), in channel catfish vaccinated with EpΔbasS and EpΔbasR against E. piscicida infection. Protection in channel catfish was associated with a significantly higher level of antibodies and upregulation of immune-related genes (IgM, IL-8 and CD8-α) in channel catfish vaccinated with EpΔbasS and EpΔbasR strains compared with non-vaccinated fish. Hybrid catfish (channel catfish ♀ × blue catfish ♂) challenges demonstrated long-term protection against subsequent challenges with E. piscicida and E. ictaluri. Our findings demonstrate BasS and BasR contribute to acid tolerance and biofilm formation, which may facilitate E. piscicida survival in harsh environments. Further, our results show that EpΔbasS and EpΔbasR mutants were safe and protective in channel catfish fingerlings, although their virulence and efficacy in hybrid catfish warrant further investigation. These data provide information regarding an important mechanism of E. piscicida virulence, and it suggests EpΔbasS and EpΔbasR strains have potential as vaccines against this emergent catfish pathogen.

Pathology and virulence of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella anguillarum in channel (Ictalurus punctatus), blue (Ictalurus furcatus), and channel × blue hybrid catfish.

In the mid-2010s, Edwardsiella tarda was reaffiliated into three discrete taxa (E. anguillarum, E. piscicida, and E. tarda), obscuring previous descriptions of E. tarda-induced pathology in fish. To clarify ambiguity regarding the pathology of E. tarda, E. piscicida, and E. anguillarum infections in US farm-raised catfish, channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and channel × blue catfish hybrids were challenged with comparable doses of each bacterium. The most severe pathology and mortality occurred in fish challenged with E. piscicida, supporting previous reports of increased pathogenicity in commercially important ictalurids, while E. anguillarum and E. tarda warrant only minimal concern. Acute pathologic lesions among bacterial species were predominantly necrotizing and characteristic of gram-negative sepsis but became progressively granulomatous over time. After 100 days, survivors were exposed to the approximate median lethal doses of E. piscicida and E. ictaluri, revealing some cross-protective effects among E. piscicida, E. anguillarum, and E. ictaluri. In contrast, no fish that survived E. tarda challenge demonstrated any protection against E. piscicida or E. ictaluri. This work supports reports of increased susceptibility of channel, blue, and hybrid catfish to E. piscicida, while highlighting potential cross-protective affects among fish associated Edwardsiella spp.