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 with catfish subjected to 12 h of hypoxia (20% oxygen saturation; 1.8 mg O2/L; 27°C) followed by 12 h of recovery in normoxia to mimic 24 h in a catfish aquaculture pond. Fish were sampled at 0-, 6-, 12-, 18-, and 24-h timepoints, with the 6- and 12-h 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 h 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.NEW & NOTEWORTHY Channel catfish are an economically important species that experience diel episodic periods of hypoxia that can reduce appetite. This is the first study to investigate their transcriptome from the hypothalamus in a simulated 24-h span in a commercial catfish pond, with 12 h of hypoxia and 12 h of normoxia. The research revealed functional groups of genes relating to hypoxia, angiogenesis, and glycolysis as well as individual target genes possibly involved in appetite regulation.

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.

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).

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.

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.

Temperature-dependent alterations in the proteome of the emergent fish pathogen Edwardsiella piscicida.

Edwardsiella piscicida is an emerging bacterial pathogen and the aetiological agent of edwardsiellosis among cultured and wild fish species globally. The increased frequency of outbreaks of this Gram-negative, facultative intracellular pathogen pose not only a threat to the aquaculture industry but also a possible foodborne/waterborne public health risk due to the ill-defined zoonotic potential. Thus, understanding the role of temperature on the virulence of this emerging pathogen is essential for comprehending the pathogenesis of piscine edwardsiellosis in the context of current warming trends associated with climate change, as well as providing insight into its zoonotic potential. In this study, significant temperature-dependent alterations in bacterial growth patterns were observed, with bacterial isolates grown at 17°C displaying higher peak growth sizes, extended lag times, and slower maximal growth rates than isolates grown at 27or 37°C. When E. piscicida isolates were grown at 37°C compared to 27 and 17°C, mass spectrometry analysis of the E. piscicida proteome revealed significant downregulation of crucial virulence proteins, such as Type VI secretion system proteins and flagellar proteins. Although in vivo models of infection are warranted, this in vitro data suggests possible temperature-associated alterations in the virulence and pathogenic potential of E. piscicida in poikilotherms and homeotherms.

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.

Lactococcus lactis MA5 is a potential autochthonous probiotic for nutrient digestibility enhancement and bacterial pathogen inhibition in hybrid catfish (Ictalurus punctatus × I. furcatus).

With the emergence of diseases, the U.S. catfish industry is under challenge. Current trends prefer autochthonous bacteria as potential probiotic candidates owing to their adaptability and capacity to effectively colonize the host's intestine, which can enhance production performance and bolster disease resistance. The objective of this study was to isolate an autochthonous bacterium as probiotic for hybrid catfish. Initially, an analysis of the intestinal microbiota of hybrid catfish reared in earthen ponds was conducted for subsequent probiotic development. Twenty lactic acid bacteria were isolated from the digesta of overperforming catfish, and most of the candidates demonstrated probiotic traits, including proteolytic and lipolytic abilities; antagonistic inhibition of catfish enteric bacterial pathogens, negative haemolytic activity and antibiotic susceptibility. Subsequent to this screening process, an isolate of Lactococcus lactis (MA5) was deemed the most promising probiotic candidate. In silico analyses were conducted, and several potential probiotic functions were predicted, including essential amino acids and vitamin synthesis. Moreover, genes for three bacteriocins, lactococcin A, enterolysin A and sactipeptide BmbF, were identified. Lastly, various protectant media for lyophilization of MA5 were assessed. These findings suggest that Lactococcus lactis MA5 can be an autochthonous probiotic from hybrid catfish, holding promise to be further tested in feeding trials.

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.

Diversity in clinical isolates of Ictalurid herpesvirus 1 (IcHV1) from U.S. farm-raised catfish and virulence assessment in channel and channel × blue catfish hybrids.

Ictalurid herpesvirus 1 (IcHV1) is the most significant viral agent in U.S. catfish aquaculture. Little is known regarding the genetic stability and antigenic variability of IcHV1. Herein, the genetic and antigenic diversity of IcHV1 field isolates was assessed by restriction fragment length polymorphism (RFLP) analysis and serum neutralization assays. RFLP analysis identified two distinct genotypes (IcHV1A and IcHV1B), both discrete from blue catfish alloherpesvirus (BCAHV). Neutralization assays with anti-IcHV1 monoclonal antibody Mab-95 indicate shared antigenic determinants for IcHV1A and IcHV1B that are absent from BCAHV, which Mab-95 did not neutralize. Virulence assessments with representative isolates demonstrate significant differences between isolates within RFLP groups and pooled RFLP group data suggest IcHV1B (pooled survival [mean ± SE]: 58.3% ± 2.6) may be more virulent than IcHV1A (survival: 68.6% ± 2.4). Rechallenges with representative IcHV1A and IcHV1B isolates indicate a cross-protective effect, with fish surviving initial exposure to IcHV1A or IcHV1B showing robust protection when subsequently re-exposed to IcHV1A or IcHV1B. This work demonstrated significant differences in virulence between case isolates, identifying two discrete IcHV1 lineages, distinct from BCAHV, with similar virulence in channel and channel × blue catfish hybrids and a cross-protective effect in catfish surviving exposure to either lineage.

Dietary supplementation of mineral nanoparticles for channel catfish (Ictalurus punctatus).

This study evaluated the supplementation of iron and copper nanoparticles in channel catfish diets and their influences on growth and health. A comparative feeding trial was carried out for 9 weeks to evaluate combinations of iron and copper nanoparticles: only iron nanoparticles (IronNP), only copper nanoparticles (CopperNP), CopperNP + IronNP, and a control diet supplemented with inorganic iron and copper (FeSO4 and CuSO4). After a 9-week feeding trial, growth performance, hematological parameters, whole-body proximate composition, and intestinal microbiota were evaluated, and fish were subjected to a bacterial challenge against Edwardsiella ictaluri to evaluate the contribution of the experimental treatments to fish health status. No statistical differences were detected for catfish fed the various diets in terms of production performance or survival after bacterial challenge. The hematocrit and RBC counts from fish fed the diet containing copper nanoparticles were significantly lower than the control group. A higher relative abundance of gram-positive bacteria was found in the digesta of catfish fed diets containing copper nanoparticles. Furthermore, in the context of hematology, iron nanoparticles did not impact the blood parameters of channel catfish; however, reduced hematocrits were observed in fish fed the copper nanoparticle diet, which lacked supplemental dietary iron, thus reinforcing the importance of dietary iron to catfish hematopoiesis. Nonetheless, additional studies are needed to investigate the effects of dietary copper nanoparticle supplementation in catfish diets to better illuminate its effects on the intestinal microbiota.

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.

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.

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.

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.

Development and efficacy of Streptococcus iniae live-attenuated vaccines in Nile tilapia, Oreochromis niloticus.

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%.

Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

AIMS: Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. METHODS AND RESULTS: The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. CONCLUSION: Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

Kudoa hypoepicardialis and associated cardiac lesions in invasive red lionfish Pterois volitans in Grenada, West Indies.

Invasive red lionfish Pterois volitans (Linnaeus, 1758) represent an ongoing ecological threat within temperate and tropical waters. Relatively little is known regarding the overall health of P. volitans and their potential for spreading pathogens in non-native regions. Lionfish collected from inshore reefs of Grenada, West Indies, in 2019 and 2021 were identified as P. volitans based on cytochrome c oxidase subunit 1 barcoding. Gross and microscopic examination of tissues revealed myxozoan plasmodia in the hearts of 24/76 (31.6%) lionfish by histopathology or wet mount cytology. Further histopathologic examination revealed severe granulomatous inflammation and myofiber necrosis associated with developing plasmodia and presporogonic life stages. Fresh myxospores were morphologically and molecularly consistent with Kudoa hypoepicardialis, being quadrate in apical view with 4 valves and 4 equal polar capsules. The spore body was 5.1-7.9 (mean: 6.0) µm long, 8.1-9.8 (8.7) µm wide, and 6.9-8.5 (7.7) µm thick. Polar capsules were 2.3-2.7 (2.5) µm long and 0.9-1.6 (1.3) µm wide. 18S small subunit rDNA sequences were 99.81-99.87% similar to sequence data from the original description of the species. Novel 28S large subunit rDNA and elongation factor 2 data, which did not match any previously reported species, were provided. This is the first account of a myxozoan parasite of P. volitans, a new host record and locality for K. hypoepicardialis, and one of few reports describing pathogen-associated lesions in invasive lionfish.

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.

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.