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The study isolated two strains of intestinal autochthonous bacteria Lactiplantibacillus plantarum1 (MH155966.1) (L1) and Lactiplantibacillus plantarum2 (MH105076.1) (L2) from the Choobdeh Abadan region. The aim of this study was to investigate the effects of different strains of probiotic bacteria on the growth performance, digestive enzyme activity, histopathologic and histomorphometric characterization of the intestine, expression of immune and growth related genes, and evaluate Lates calcarifer resistance against Vibrio alginolyticus. To achieve this, for each treatment 60 L. calcarifer juveniles (75 ± 12 g) were randomly distributed in three fiberglass tanks (300 L) and fed for 45 days. The treatments were established as Diet 1 (control diet); L1 (diet with Lb. plantarum isolated 1); L2 (diet with Lb. plantarum isolated 2) with a bacterial concentration of 1 × 10 9 CFU/g. Nine fish from each treatment were sampled and examined, after euthanasia. The fish were placed 2 cm from the beginning of the intestine for microscopic sampling of villi height, villi width and thickness of the epithelium, with 3 treatments: The result showed differences in the mean values of total weight were found at the end of the experiment. After 45 days of culture, the fish fed with L1 had higher (P < 0.05) growth performance than the other treatment groups. But at the end of the trial, in L2, the digestive enzyme activities were higher (P < 0.05) than the other treatment groups. The fishes fed diets supplemented with the L2 group, like the digestive enzyme activities test, presented an increase in the thickness of the epithelium of the intestine, and villus height, and villus width were greatest in L2. Fish feeding with L1 and L2 probiotics induced higher transcription levels of interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor (EGF), and Transforming Growth Factor Beta (TGF-β) genes in the gut, which may correlate with better immune and hematological parameters in these groups. The results of the challenge test revealed that the percentage of survival was significantly higher in L1 (76.2%) and L2 (80.95%) treatments than in the control (P < 0.05). These results indicate that host-derived probiotics (Lb. plantarum) have significant potential as important probiotics to enhance nutrient utilization, Digestive enzymes, and metabolism by increasing the gut surface area of Lates calcarifer juveniles at 45 days of culture., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2024 John Wiley & Sons Ltd.)

Objective: We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a "fit for purpose" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc., Methods: Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays., Result: The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay "gold standard" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book., Conclusion: The "fit for purpose" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc., (© 2024 American Fisheries Society.)

There is a claimed increase in the global prevalence and incidence of emerging diseases observed in many organisms. Myxozoa represents an essential group of metazoan parasites that hold both economic and ecological significance. In the current study, 1% of the fish population at two commercial goldfish (Carassius auratus) farms in Tehran and Ghom province, Iran, developed cavitating muscular lesions resembling humps in February 2021 and January 2022. Fish displaying pathological abnormalities were transported to the Ornamental Fish Clinic and subjected to clinical examination. Light microscopy was subsequently used to examine wet smears of skin and gills, as well as whitish exudate. In addition, tissue homogenates were collected for more precise identification and molecular confirmation. The study discovered that individuals from the goldfish farms were infected with the pathogenic myxozoan Myxobolus lentisuturalis, which caused significant damage to the epaxial muscles. The spores collected from the humps had a lack of uniformity and were primarily ellipsoidal in shape. Histopathological analysis also revealed parasites in various stages of development, such as plasmodia and spores, as well as inflammatory cell infiltration (macrophage, giant cell and lymphoplasmacytic infiltration) between skeletal muscle fibers. Phylogenetic analysis of M. lentisuturalis was performed by using MEGA 11 and the maximum likelihood method. M. lentisuturalis is a myxozoan parasite that has been sparsely recorded and lacks widespread recognition. The current study is the first clinical, histopathological, and molecular characterization of M. lentisuturalis isolated from the skeletal musculature of goldfish (C. auratus) in Iran., (© 2024. The Author(s).)

The current research aimed to shed light on the efficacy of Escherichia coli strain Nissle 1917 (EcN) on goldfish (……) growth, gut immunity, morphology, bacterial nutritional enzyme activity and resistance to Aeromonas hydrophila infection. Fish fed with EcN at 10 6 , 10 7 and 10 8  CFU/g feed for 80 days showed an enhancement in growth better than control fish. The gut innate immunity in terms of lysozyme activity, immunoglobulin and total protein levels was increased in the treatment fish with the best result being observed in fish fed EcN at 10 8  CFU/ g. In addition, an increase was noted in the upregulation of immune-relevant genes, namely lysozyme, interleukin-1β, inducible nitric oxide synthase and tumor necrosis factor α of fish intestine. A marked surge in the number of proteolytic and heterotrophic bacteria was noted in the gut of fish nourished with the probiotic. Histological studies exhibited an improvement in the intestinal absorption surface area, intraepithelial lymphocyte count and goblet cell density. Significantly higher survival rate was obtained in fish fed EcN at 10 8  CFU/g compared with the fish fed with the basal diet. These data exhibited the beneficial effect of EcN on goldfish growth, digestive enzymes, intestine heterotrophic bacteria and resistance against Aeromonas hydrophila challenge. This study confirmed the favorable outcomes resulting from the administration of EcN at10 8 CFU/g., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2024 John Wiley & Sons Ltd.)

Spring viremia of carp virus (SVCV) is a rhabdovirus that primarily infects cyprinid finfishes and causes a disease notifiable to the World Organization for Animal Health. Amphibians, which are sympatric with cyprinids in freshwater ecosystems, are considered non-permissive hosts of rhabdoviruses. The potential host range expansion of SVCV in an atypical host species was evaluated by testing the susceptibility of amphibians native to the Pacific Northwest. Larval long-toed salamanders Ambystoma macrodactylum and Pacific tree frog Pseudacris regilla tadpoles were exposed to SVCV strains from genotypes Ia, Ib, Ic, or Id by either intraperitoneal injection, immersion, or cohabitation with virus-infected koi Cyprinus rubrofuscus . Cumulative mortality was 100% for salamanders injected with SVCV, 98-100% for tadpoles exposed to virus via immersion, and 0-100% for tadpoles cohabited with SVCV-infected koi. Many of the animals that died exhibited clinical signs of disease and SVCV RNA was found by in situ hybridization in tissue sections of immersion-exposed tadpoles, particularly in the cells of the gastrointestinal tract and liver. SVCV was also detected by plaque assay and RT-qPCR testing in both amphibian species regardless of the virus exposure method, and viable virus was detected up to 28 days after initial exposure. Recovery of infectious virus from naïve tadpoles cohabited with SVCV-infected koi further demonstrated that SVCV transmission can occur between classes of ectothermic vertebrates. Collectively, these results indicated that SVCV, a fish rhabdovirus, can be transmitted to and cause lethal disease in two amphibian species. Therefore, members of all five of the major vertebrate groups (mammals, birds, reptiles, fish, and amphibians) appear to be vulnerable to rhabdovirus infections. Future research studying potential spillover and spillback infections of aquatic rhabdoviruses between foreign and domestic amphibian and fish species will provide insights into the stressors driving novel interclass virus transmission events.

Eustrongylides excisus is a fish-borne zoonotic parasite known to infect various fish species, including Northern pike (Esox Lucius). This nematode, belonging to the family Dioctophymatidae, has a complex life cycle involving multiple hosts. This study aimed to investigate the occurrence of Eustrongylides nematodes in Northern pike (E. Lucius) collected from Mijran Dam (Ramsar, Iran). Between June and October 2023, an investigation was conducted on Northern pike from Mijran Dam in Ramsar, Iran, following reports of reddish parasites in their muscle tissues. Sixty fish were examined at the University of Tehran, revealing live parasites in the muscles, which were then analyzed microscopically and preserved for a multidisciplinary study. The skeletal muscle tissues of 85% (51/60) of fish specimens were infected by grossly visible larvae which were microscopically identified as Eustrongylides spp. In histopathological examination, the lesion was composed of encapsulated parasitic granulomatous myositis. Microscopically, the cystic parasitic granulomas compressed the adjacent muscle fibers, leading to their atrophy and Zenker's necrosis. Moreover, epithelioid macrophages, giant cells and mononuclear inflammatory cells were present around the larvae and between the muscle fibers. Finally, a molecular analysis by examining the ITS gene region, revealed that they belong to the species E. excisus. Eustrongylidiasis in northern Iran necessitates further research into the biology, epidemiology, and control of Eustrongylides nematodes, focusing on various hosts. This study is the first to comprehensively characterize E. excisus in Northern pike in Ramsar, Iran, raising concerns about possible zoonotic transmission., (© 2024. The Author(s).)

Infectious diseases are a leading cause of losses in the aquaculture industry and conservation programs globally. Simultaneously, infectious diseases pose a substantial risk to fish being hatchery-reared and released into natural habitats for conservation purposes, including the Great Lakes lake sturgeon ( Acipenser fulvescens , i.e., GL-LST). Recently, an alloherpesvirus (lake sturgeon herpesvirus 2, i.e., LSHV-2) capable of inducing disease and/or mortality in adult and juvenile GL-LSTs was detected in two adult GL-LST populations. To begin developing disease prevention and/or control methods, in vitro experiments were designed to determine the susceptibility of LSHV-2 to disinfectants commonly used in hatchery and aquaculture facilities (Virkon ® -Aquatic: potassium peroxymonosulfate; Ovadine ® : polyvinylpyrrolidone iodine complex; and Perox-Aid ® : hydrogen peroxide). Cultured LSHV-2 was exposed to each disinfectant at two concentrations (Virkon ® -Aquatic: 0.5% and 1%; Ovadine ® : 50 and 100 ppm; and Perox-Aid ® : 500 and 1000 ppm) in duplicate for durations of 1, 10, and 30 min. Following exposure, the disinfectant was neutralized, and after a 14-day incubation period on a white sturgeon × lake sturgeon hybrid cell line (WSxLS), percent reduction was calculated by comparing the 50% tissue culture infectious doses (TCID 50 /mL) of the virus with and without disinfectant exposure. When exposed to Perox-Aid ® , LSHV-2 percent reduction ranged from 58.7% to 99.5%. When exposed to Ovadine ® , the percent reduction ranged from 99.4% to 100%. Lastly, the percent reduction when exposed to Virkon ® -Aquatic was 100% for both concentrations and all timepoints. The results herein provide evidence that both Virkon ® -Aquatic and Ovadine ® are virucidal to LSHV-2 and may represent a means to reduce virus transmission risk under field settings.

A novel microsporidium was observed in wild swamp guppies Micropoecilia picta from Levera Pond within Levera National Park Grenada, West Indies. Initial observations indicated similarity with Pseudoloma neurophilia, an important pathogen in zebrafish Danio rerio. P. neurophilia exhibit broad host specifity, including members of the family Poecillidae, and both parasites infect the central nervous system. However, spore morphology and molecular phylogeny based on rDNA showed that the swamp guppy microsporidium (SGM) is distinct from P. neurophilia and related microsporidia (Microsporidium cerebralis and M. luceopercae). Spores of the SGM were smaller than others in the clade (3.6 µm long). Differences were also noted in histology; the SGM formed large aggregates of spores within neural tissues along with a high incidence of numerous smaller aggregates and single spores within the surface tissue along the ventricular spaces that extended submeninx, whereas P. neurophilia and M. cerebralis infect deep into the neuropile and cause associated lesions. Analysis of small subunit ribosomal DNA sequences showed that the SGM was <93% similar to these related microsporidia. Nevertheless, one of 2 commonly used PCR tests for P. neurophilia cross reacted with tissues infected with SGM. These data suggest that there could be other related microsporidia capable of infecting zebrafish and other laboratory fishes that are not being detected by these highly specific assays. Consequently, exclusive use of these PCR tests may not accurately diagnose other related microsporidia infecting animals in laboratory and ornamental fish facilities.

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., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., (© 2023 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

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., (© 2023 John Wiley & Sons Ltd.)

Purpose: The presence of Blastocystis sp. is commonly observed in humans and different animals, displaying a wide range of genetic variations with the discovery of multiple subtypes (STs). However, the prevalence and distribution of these STs in edible marine fish and marine mammals remain uncertain. This study marks the first survey conducted in Iran and the second global molecular investigation to examine the occurrence and STs distribution of Blastocystis in various species of edible marine fish., Methods: This study screened 200 fresh intestinal contents from 10 well-known fish species (Narrow-barred mackerel, Indo-pacific king mackerel, Tigertooth croaker, Silver pomfret, Black pomfret, Longtail tuna, John's snapper, Blackspotted croaker, Four-finger threadfin, and Javelin grunter) in southern Iran, caught in the Persian Gulf. All collected samples were evaluated by microscopy and SSU-PCR methods., Results: Based on both microscopy and PCR, the overall prevalence of Blastocystis sp. in evaluated fish species was 2% (4/200). In brief, Blastocystis sp. was reported from Narrow-barred mackerel [10% (2/20)], Silver pomfret [5% (1/20)], and Tigertooth croaker [5% (1/20)]. Interestingly, among infected fish species three zoonotic STs (ST1, ST2, and ST7) were identified. ST2 was the most predominant ST [50% (2/4)], followed by ST1 and ST7, one sample each [5% (1/20)]., Conclusion: Overall, the prevalence and STs distribution of Blastocystis in edible marine fish along with the possibility of its zoonotic transmission are still open to question and require extensive and more detailed studies., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., (© 2023 American Fisheries Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and is responsible for substantial losses in farm and hatchery-reared salmonids (Family Salmonidae ). Although F. psychrophilum infects multiple economically important salmonids and is transmitted horizontally, the extent of knowledge regarding F. psychrophilum shedding rates and duration is limited to rainbow trout ( Oncorhynchus mykiss ). Concurrently, hundreds of F. psychrophilum sequence types (STs) have been described using multilocus sequence typing (MLST), and evidence suggests that some variants have distinct phenotypes, including differences in host associations. Whether shedding dynamics differ among F. psychrophilum variants and/or salmonids remains unknown. Thus, three F. psychrophilum isolates (e.g., US19, US62, and US87) in three MLST STs (e.g., ST13, ST277, and ST275) with apparent host associations for coho salmon ( O. kisutch ), Atlantic salmon ( Salmo salar ), or rainbow trout were intramuscularly injected into each respective fish species. Shedding rates of live and dead fish were determined by quantifying F. psychrophilum loads in water via quantitative PCR. Both live and dead Atlantic and coho salmon shed F. psychrophilum , as did live and dead rainbow trout. Regardless of salmonid species, dead fish shed F. psychrophilum at higher rates (e.g., up to ~10 8 -10 10 cells/fish/hour) compared to live fish (up to ~10 7 -10 9 cells/fish/hour) and for a longer duration (5-35 days vs 98 days); however, shedding dynamics varied by F. psychrophilum variant and/or host species, a matter that may complicate BCWD management. Findings herein expand knowledge on F. psychrophilum shedding dynamics across multiple salmonid species and can be used to inform future BCWD management strategies.IMPORTANCE Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, both of which cause substantial losses in farmed and hatchery-reared salmon and trout populations worldwide. This study provides insight into F. psychrophilum shedding dynamics in rainbow trout ( Oncorhynchus mykiss ) and, for the first time, coho salmon ( O. kisutch ) and Atlantic salmon ( Salmo salar ). Findings revealed that live and dead fish of all fish species shed the bacterium. However, dead fish shed F. psychrophilum at higher rates than living fish, emphasizing the importance of removing dead fish in farms and hatcheries. Furthermore, shedding dynamics may differ according to F. psychrophilum genetic variant and/or fish species, a matter that may complicate BCWD management. Overall, study results provide deeper insight into F. psychrophilum shedding dynamics and will guide future BCWD management strategies., Competing Interests: The authors declare no conflict of interest.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, causes substantial economic losses in salmonid farms and hatcheries. Some multilocus sequence types (ST) of F. psychrophilum are more likely to be associated with fish farms and hatcheries, but it is unclear if these patterns of association represent genetic lineages that are more adapted to aquaculture environments. Towards elucidating the disease ecology of F. psychrophilum, the culturability of 10 distinct F. psychrophilum STs was evaluated for 13 weeks in three microcosms including sterilized well water, sterilized well water with commercial trout feed, or sterilized well water with raceway detritus. All STs remained culturable in each of the microcosms for at least 8 weeks, with bacterial concentrations often highest in the presence of raceway detritus. In addition, most (e.g., 90%) STs remained culturable for at least 13-weeks. Significant differences in log 10 cfus were observed among STs, both within and between microcosms, suggesting potential variability in environmental persistence capacity among specific variants. Collectively, results highlight the ability of F. psychrophilum to not only persist for weeks under nutrient-limited conditions but also thrive in the presence of organic substrates common in fish farms and hatchery-rearing units., (© 2024 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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., (© 2023 American Fisheries Society.)

Flavobacterial diseases, caused by bacteria in the order Flavobacteriales, are responsible for devastating losses in farmed and wild fish populations worldwide. The genera Flavobacterium (Family Flavobacteriaceae ) and Chryseobacterium ( Weeksellaceae ) encompass the most well-known agents of fish disease in the order, but the full extent of piscine-pathogenic species within these diverse groups is unresolved, and likely underappreciated. To identify emerging agents of flavobacterial disease in US aquaculture, 183 presumptive Flavobacterium and Chryseobacterium isolates were collected from clinically affected fish representing 19 host types, from across six western states. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis using the gyrB gene. Antimicrobial susceptibility profiles were compared between representatives from each major phylogenetic clade. Of the isolates, 52 were identified as Chryseobacterium species and 131 as Flavobacterium . The majority of Chryseobacterium isolates fell into six clades (A-F) consisting of ≥ 5 fish isolates with ≥ 70% bootstrap support, and Flavobacterium into nine (A-I). Phylogenetic clades showed distinct patterns in antimicrobial susceptibility. Two Chryseobacterium clades (F & G), and four Flavobacterium clades (B, G-I) had comparably high minimal inhibitory concentrations (MICs) for 11/18 antimicrobials tested. Multiple clades in both genera exhibited MICs surpassing the established F. psychrophilum breakpoints for oxytetracycline and florfenicol, indicating potential resistance to two of the three antimicrobials approved for use in finfish aquaculture. Further work to investigate the virulence and antigenic diversity of these genetic groups will improve our understanding of flavobacterial disease, with applications for treatment and vaccination strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Heckman, Yazdi, Pomaranski, Sebastião, Mukkatira, Vuglar, Cain, Loch and Soto.)

Pseudomurraytrema fergusoni n. sp. is described from the Pealip Redhorse, Moxostoma pisolabrum from the Black River (White River drainage), Lawrence County, Arkansas, USA. This represents the second monogenean described from M. pisolabrum as well as the second species of Pseudomurraytrema reported from an Arkansas catostomid. The description includes partial 18S rRNA and 28S rRNA gene sequences (732 bp and 851 bp, respectively), helping fill a void in sequence data from North American monogeneans, particularly those in the genus Pseudomurraytrema. In addition, histopathologic changes associated with the infection resulted in severe localized pathologic lesions in gills of the host, suggesting compromise of respiratory surfaces within affected areas adjacent to the worms., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: Carnobacterium maltaromaticum is considered an emerging pathogen of salmonids in the United States and around the world., Methods: Bacterial cultures obtained from the posterior kidney and skin of moribund Rainbow Trout Oncorhynchus mykiss from a commercial aquaculture facility in Virginia, USA, grew C. maltaromaticum, which was confirmed by additional phenotypic and molecular characterization., Result: A presumptive diagnosis based on the clinical signs, necropsy observations, histopathology, and bacterial cultures was bacterial septicemia due to C. maltaromaticum., Conclusion: This represents the first documentation of C. maltaromaticum in Rainbow Trout from Virginia., (© 2023 The Authors. Journal of Aquatic Animal Health published by Wiley Periodicals LLC on behalf of American Fisheries Society.)

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., (© 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Enteric septicemia of catfish, which is caused by Edwardsiella ictaluri, is detrimental to farmed Channel Catfish Ictalurus punctatus. The hemin receptor HemR is involved in binding and uptake of heme into bacteria. Here, we explored pathological and ultrastructural changes in catfish fry that were immunized with a triple hemR mutant of E. ictaluri and challenged with wild-type E. ictaluri (EiWT) 28 d after immunization. Following immunization, pathological changes in the triple hemR-immunized fry were less severe compared to the EiWT-exposed control fry. Widely disseminated bacteria and severe necrosis in most organs, especially the kidney and spleen, were detected in both groups at days 4, 5, and 6. Multifocal granulomatous encephalitis with bacteria was seen in hemR-immunized fry at days 21 and 28 and in EiWT-exposed control fry at day 14. Phagocytic cells in the kidney and spleen of EiWT-exposed control fry contained more replicating bacteria compared to hemR-immunized fry. During the EiWT challenge of immunized fry, a robust immune response was observed in the triple hemR-immunized fry compared to the sham-vaccinated group. Many activated phagocytic cells were detected in the kidney and spleen with fragmented or no bacteria in the triple hemR-immunized fry. Our data suggested that virulence of triple hemR was lower and the onset of the lesions was delayed compared to EiWT. Additionally, triple hemR-immunized fry could mount an immune response and had milder lesions compared to the sham control after EiWT exposure., (© 2022 American Fisheries Society.)

During October and November 2021, 33 creek chubs, Semotilus atromaculatus, were collected from 3 sites in Polk County, Arkansas (Ouachita River drainage), and their gills, gallbladder, fins, integument, musculature, and other major organs were examined for myxozoans. The gills of 9 (27%) were infected with a new myxozoan, Myxobolus fountainae n. sp. Qualitative and quantitative morphological data were from fresh and formalin-fixed preserved spores, while molecular data consisted of a 1918 base pair sequence of the partial small subunit ribosomal RNA gene. Phylogenetic analysis grouped M. fountainae n. sp. with the other leuciscid-infecting myxobolids from North America and within a larger clade of European myxozoans. In addition, histological information is provided on the infection. A previous record of Myxobolus muelleriBütschli, 1882, from the gills and ureters of S. atromaculatus is considered invalid and represents an unknown species. Myxobolus fountainae n. sp. is the only named myxozoan known to infect the gill filaments of S. atromaculatus, whereas Myxobolus pendula (Guilford, 1967) infects the gill arches., (© American Society of Parasitologists 2022.)

Flavobacterium psychrophilum , the etiological agent of bacterial coldwater disease and rainbow trout fry syndrome, causes considerable losses in salmonid aquaculture globally. Systemic F. psychrophilum infections in rainbow trout ( Oncorhynchus mykiss ) lead to a range of clinical signs, including ulcerative lesions in the skin and muscle and splenitis. Previous studies offered an integrative analysis of the skeletal muscle response to F. psychrophilum infection in rainbow trout. However, little is known about the molecular mechanism of immune response in the spleen, which is an important immune organ of rainbow trout. Here, we investigated the time-course splenic transcriptome profiles in uninfected rainbow trout (CK) and F. psychrophilum -infected rainbow trout at day 3 and day 7 (D3, D7) by RNA-seq analyses. Among the 7,170 differentially expressed genes (DEGs) in the three comparisons (D3 vs. CK, D7 vs. CK, D3 vs. D7), 1,286 DEGs showed consistent upregulation or downregulation at D3 and D7 and were associated with pattern recognition, acute-phase response, complement cascade, chemokine and cytokine signaling, and apoptosis. The Real time quantitative PCR (RT-qPCR) analysis of eight DEGs confirmed the accuracy of the RNA-Sequencing (RNA-seq) data. Our results reflected a general process from pathogen recognition to inflammatory cytokine generation and delineated a putative Toll-like receptor signaling pathway in rainbow trout spleen, following F. psychrophilum infection. Taken together, these results provide new insights into the molecular mechanism of the immune response to F. psychrophilum infection and are a valuable resource for future research on the prevention and control of bacterial coldwater disease during salmon culture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Deng, Wang, Loch, Chen, Lu, Cao, Fan and Li.)

Constructs bearing the cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (β-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F 3 channel catfish family transgenic for cecropin transgene driven by the CMV promoter, and one F 1 channel catfish family transgenic for cecropin transgene driven by the common carp β-actin promoter were produced. F 3 and F 1 individuals exhibited enhanced disease resistance when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). Inheritance of the transgene by the F 1 and F 3 generation was 15% and 60%, respectively. Growth rates of the cecropin transgenic and non-transgenic full siblings (controls) channel catfish were not different (P > 0.05). All transgenic fish showed significant resistance to infection by ESC at day 3 and day 4 post exposure (P = 0.005). No correlation was detected between body weight and time to death for all genetic groups (P = 0.34). Results of our study confirmed that genetic enhancement of E. ictaluri resistance can be achieved by cecropin transgenesis in channel catfish. In addition to survival rate, improving survival time is essential because the extension of survival time gives a better chance to apply treatments to stop the bacterial infection., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Edwardsiella piscicida is a growing problem for catfish aquaculture in the southeastern United States, particularly in channel (Ictalurus punctatus) x blue (I. furcatus) catfish hybrids. Research has shown E. piscicida isolates recovered from farmed catfish in Mississippi form at least five discrete phyletic groups, with no apparent differences in virulence in channel and hybrid catfish. Laboratory trials have shown a live-attenuated E. ictaluri vaccine (340X2) cross-protects against at least one E. piscicida isolate (S11-285) in channel and hybrid catfish, although it is unknown if this protection exists for other E. piscicida variants. To this end, channel and hybrid catfish were immunized by immersion with E. ictaluri 340X2. Thirty days later, fish were challenged by intracoelomic injection with representative E. piscicida variants from each phyletic group. Relative percent survival (RPS) for hybrids ranged from 54.7% to 77.8%, while RPS in channels ranged from 80.5% to 100%. A second study investigated whether channel and hybrid catfish exposed to heterologous E. piscicida isolates were similarly protected against wild-type E. ictaluri. Fish were exposed by bath immersion to representative E. piscicida isolates from each phyletic group. Thirty days post-immunization, fish were challenged by immersion with wild-type E. ictaluri isolate S97-773. Regardless of variant, previous exposure to heterologous E. piscicida isolates significantly improved survival following E. ictaluri challenge. These findings suggest the presence of shared and conserved antigens among E. piscicida and E. ictaluri that could be exploited by application of polyvalent or cross-protective vaccines., (© 2022 John Wiley & Sons Ltd.)

Lake whitefish (Coregonus clupeaformis; LWF) is an economically and ecologically valuable native species to the Great Lakes, but recent declines in their recruitment have generated significant concern about their future viability. Although studies have sought to identify factors contributing to declining recruitment, the potential role(s) of infectious diseases has not been thoroughly investigated. In 2018 and 2019, adult LWF were collected from Lakes Superior, Michigan, and Huron for clinical examination and bacteriological analyses. Herein, we describe the first isolation of Flavobacterium psychrophilum, aetiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), from systemically infected adult LWF. Bacterial isolates were yellow-orange, Gram-negative, filamentous bacilli that were oxidase and catalase positive, and produced a flexirubin-type pigment in 3% potassium hydroxide. Isolate identity was confirmed via F. psychrophilum-specific PCR, and multilocus sequence typing revealed three new singleton sequence types (STs) that were distinct from all previously described F. psychrophilum STs. The prevalence of F. psychrophilum infections was 3.3, 1.7, and 0.0% in Lakes Superior, Michigan and Huron respectively. Findings illustrate the potential for F. psychrophilum to cause systemic infections in adult LWF and highlight the need for future studies to investigate the bacterium's potential role in declining LWF recruitment., (© 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

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., (© 2022 Society for Applied Microbiology.)

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria., (© 2022 John Wiley & Sons Ltd.)

Previously, rainbow trout fed deoxynivalenol (DON) or partially fed (pair-fed) for 4 weeks before and during experimental infection with Flavobacterium psychrophilum had significantly decreased mortality rates. Similar results were obtained in the present study after 12 days, but not after 6 days, feeding 5 ppm DON or pair-fed before infection. Furthermore, feeding 250 ppm chloroquine (CQ) also reduced mortality (p = .052) compared with controls and may have promise for treatment of some fish disease. Parallel groups of fish were maintained on the respective treatments for 15 days, with an additional group that was fasted, but were not infected to monitor autophagy. Fish that were fasted or fed DON had significantly increased LC3II in the liver and fasted fish had significantly decreased LC3II in muscle compared with controls using western blot. There was no difference in LC3II signal in the spleen of any treatment group. Fish that were fasted or pair-fed had significant up-regulation of the Atg genes atg4, atg7, lc3, gabarap and atg12 in muscle using quantitative PCR. Less alteration of Atg expression was seen in liver. Fish treated with CQ had significantly increased expression of atg4, becn1, lc3 and atg12 in the liver. Fish fed DON for 15 days had few alterations of Atg genes in either the liver or muscle. It is still not clear if autophagy is responsible for the resistance of rainbow trout fed DON, CQ or pair-fed before F. psychrophilum infection., (© 2022 John Wiley & Sons Ltd.)

Advances in hybridization practices in U.S. catfish aquaculture have led to increased production of channel (Ictalurus punctatus) × blue catfish (Ictalurus furcatus) hybrids to capitalize on their more favorable production characteristics. However, the effects of typical channel catfish pathogens on hybrids are not well understood, including the digenean Bolbophorus damnificus, which has caused significant losses in channel catfish production. Three experiments were conducted to assess the longevity and site specificity of 2 life stages of B. damnificus impacting catfish production. The first experiment investigated the cercarial longevity and infectivity of B. damnificus over time. Channel catfish were individually challenged with 100 cercariae/fish with cercariae aged in 12-hr time intervals over 5 days (n = 5 fish/time point), with metacercarial cysts excised and enumerated 14 days postchallenge. There was a decrease in cercaria viability and encysted metacercariae over the first 36 hr, with the 12-hr time point having both the greatest cercaria survival and the highest number of metacercariae in exposed fish. The second experiment investigated the longevity of metacercariae within both channel and hybrid catfish. Fish (n = 30) were exposed to 2 treatments (75 or 150 cercariae/fish), and 2 fish from each treatment were sampled every 3 mo for 13 mo. Live metacercariae, based on motility observed after excystment, were found in both species up to 13 mo postchallenge, indicating the metacercariae of B. damnificus can persist throughout an entire growing season in both channel and hybrid catfish. The third experiment investigated the site specificity of metacercariae within both channel and hybrid catfish. Fish (n = 60/species) were challenged with 300 cercariae/fish and 9 fish/species were sampled after 90 days. Metacercariae were excised and enumerated from the anterior midsection (head and body), posterior midsection (trunk/caudal peduncle), ventral (belly), and caudal fin (tail) sections of each fish. Overall, the trunk/caudal peduncle had a 2-fold increase in the number of metacercariae excised, and although not significantly higher, results indicate this region should be the focal point of pondside assessment for the presence of B. damnificus because of ease of detection of encysted metacercariae., (© American Society of Parasitologists 2022.)

An abundance of morphologically variable Henneguya species complicates the understanding of disease relationships between ictalurid catfish and myxozoan (Phylum: Cnidaria) parasites on North American aquaculture operations. Henneguya ictaluri, the cause of proliferative gill disease (PGD) in channel and hybrid catfish, is arguably the most important parasite of commercial catfish aquaculture in the southeastern United States. While research indicates arrested development and limited sporogenesis of H. ictaluri in channel (Ictalurus punctatus) × blue (Ictalurus furcatus) hybrid catfish, incidents of PGD persist in hybrid production systems. This work investigated the influence of fish host on myxozoan community composition and diversity within naturally infected gill tissues from diagnostic case submissions to the Aquatic Research and Diagnostic Laboratory in Stoneville, Mississippi, from 2017 to 2019. Gills collected from farm-raised catfish with clinical PGD were subjected to metagenomic amplicon sequencing of the myxozoan 18S SSU rDNA gene diagnostic variable region 3 (DVR3). Myxozoan community composition significantly differed between channel and hybrid catfish PGD cases, with channel catfish having more diverse community structures. Channel catfish gills had a greater relative abundance of H. ictaluri in 2017 and 2019, while no differences were observed in 2018. Importantly, H. ictaluri was present in all channel and hybrid catfish PGD cases across all years; however, H. ictaluri was not the most abundant myxozoan in almost half the cases examined, suggesting other myxozoan species may also contribute to PGD pathology. The detection of numerous known and unclassified myxozoan sequences in addition to H. ictaluri provides evidence PGD may involve mixed species infections. Furthermore, the presence of numerous unclassified myxozoan sequences in gill samples from clinical PGD cases indicates the number of described species from U.S. farm-raised catfish vastly underestimates the true myxozoan diversity present within the varied pond microcosms associated with catfish aquaculture., (© American Society of Parasitologists 2022.)

Previous morphological and histological data are supplemented with molecular and ultrastructural data for a Henneguya sp. isolated from farm-raised channel catfish Ictalurus punctatus in Mississippi, USA. Myxospores were cryptic, encapsulated within a thin layer of epithelium in the gill lamellae with spore measurements consistent with the original description of Henneguya postexilis Minchew, 1977. Myxospores were 42.7-49.1 µm in total length with spore bodies 12.1-17.2 × 3.6-4.8 × 2.9-3 µm. Polar capsules were of unequal length, with the longer capsule being 4.4-6.7 × 1.1-1.6 µm and the shorter capsule being 4.4-6.4 × 1.1-1.6 µm. Polar tubules had 6-8 turns. Caudal processes were 25.7-38.1 µm in length. Spores were encapsulated in a thin layer of epithelium in the gill lamellae. Molecular data from the most commonly used markers for myxozoan identification and phylogeny, partial 18S small subunit ribosomal gene (SSU), partial 28S large subunit ribosomal gene (LSU), and elongation factor 2 (EF2) were generated for H. postexilis. Additionally, novel data for LSU and EF2 were generated for archived myxozoan specimens from farm-raised catfish (H. mississippiensis, H. ictaluri, H. exilis, H. adiposa, H. sutherlandi, H. bulbosus, Unicauda fimbrethilae), as well as archived specimens from wild fish (H. laseeae [from Pylodictis olivaris], Hennegoides flockae [from Aphredoderus sayanus], Myxobolus cloutmani [from Cycleptus elongatus]. These include the first EF2 sequence data for the genera Hennegoides and Unicauda. Phylogenetic analyses using these data placed H. postexilis in well supported clades with other ictalurid-infecting Henneguya species. Phylogenetic signal assessments on these analyses suggest that while SSU provided the greatest phylogenetic signal, LSU yielded comparable signal, supporting previous work implying this region may be underutilised in reconstructing myxobolid phylogenies., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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%., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Copper and vitamin C are micronutrients needed for the living organism's functions. Vitamin C has a great effect on the immune system of fish. The present study aimed to evaluate the effects of dietary copper nanoparticles (Cu-NPs) and vitamin C (VC) supplementations on rainbow trout (Oncorhynchus mykiss) juveniles. So, 216 rainbow trout juveniles were randomly assigned to six groups with trial diets supplemented with Cu-NPs and VC including 0/0 (T1, control diet), 0/250 (T2), 0/500 (T3), 2/250 (T4), 2/500 (T5), and 2/0 (T6) mg Cu-NPs/VC per kg diet. After the feeding trial for 60 days, the fish were challenged with Yersinia ruckeri, and the survival rate was calculated for 15 days. Based on the data analysis, weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER), lysozyme, alternative complement activity (ACH50), hematocrit (Hct), hemoglobin (Hb), and mean corpuscular volume (MCV) were significantly (p < 0.05) increased in the fish fed on T4 and T5 diets compared with the control group. Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) were significantly (p < 0.05) decreased in the fish fed with diets contain Cu-NPs and VC (T4 and T5). The expressions of TNF-α, IL-1ß, IL-10, SOD, CAT, and GPX genes were significantly (p < 0.05) decreased in the fish fed on T3, T4, and T5 diets versus the control. In addition, the dietary Cu-NPs and VC supplementations significantly enhanced resistance against pathogens and led to the control of infection in rainbow trout. In conclusion, Cu-NPs and VC administered as feed additives at 2/250-500 mg/kg elevated the growth performance, antioxidant capacity, and health of rainbow trout., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Indiscriminate antimicrobial use (AMU) in aquaculture to treat and prevent diseases is common and can lead to the emergence of antimicrobial-resistant micro-organisms, potentially impacting public health and connected ecosystems. This study aimed to develop a typology to classify and characterise interventions to reduce AMU in aquaculture and identify points of action. Seventeen aquaculture and animal health professionals in Asian and African countries were interviewed to gather information on characteristics of interventions in different contexts to develop a typology. Seven types of interventions were defined: (i) legislation and regulations; (ii) industry rules and standards; (iii) voluntary instruments; (iv) commercial technology and alternatives to antimicrobials; (v) on-farm management; (vi) learning and awareness-raising; and (vii) activities with co-benefits. Types were based on intervention function, scope of implementation, implementer, compulsion, strength of the intervention, AMU/antimicrobial resistance (AMR) objective and stakeholder to influence. For each type, examples were described and discussed. The most common interventions to address AMU and AMR were legislative and regulatory frameworks and voluntary instruments, including National Action Plans. Interventions addressing AMU/AMR specifically were scarce. Other interventions focused on indirect effect pathways to AMU and AMR reduction aiming to improve good aquaculture practices, disease prevention and improved management. Monitoring and evaluation of these interventions were found to be rare, only present for interventions driven by development projects and international agencies. The presented typology of existing strategies and interventions addressing AMU/AMR in aquaculture systems can guide evaluation of AMR-sensitive interventions that promote responsible AMU, and informs the design and implementation of future interventions., Competing Interests: Competing interests None declared., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Edwardsiella piscicida is an emergent global fish pathogen with a wide host range, although host associations driving genetic diversity remain unclear. This study investigated the genetic and virulence diversity of 37 E. piscicida isolates recovered from 10 fish species in North America. Multilocus sequence analysis (MLSA) was conducted using concatenated alignments of the gyrB, pgi and phoU sequences. MLSA clustered the tested isolates into six discrete clades. In light of recent disease outbreaks in cultured salmonids, the virulence of each clade was evaluated in Chinook salmon Oncorhynchus tshawytscha fingerlings following intracoelomic challenge of ~10 6  CFU/fish. Challenged and control fish were monitored for 21d, and microbiological and histological examination was performed on dead and surviving fish. Peak mortality occurred 3-5 days post-challenge (dpc) regardless of isolate or genetic group. Edwardsiella piscicida was recovered from all moribund and dead animals. At 21 dpc, fish challenged with isolates from clades II, III and IV presented cumulative mortality ≥83.3%, whereas isolates from clade I, V and VI resulted in cumulative mortality ≤71.4%. This study suggests an underlying genetic basis for strain virulence and potential host associations. Further investigations using other fish models and variable challenge conditions are warranted., (© 2021 John Wiley & Sons Ltd.)

The bacterium Edwardsiella piscicida causes significant losses in global aquaculture, particularly channel (Ictalurus punctatus) × blue (I. furcatus) hybrid catfish cultured in the south-eastern United States. Emergence of E. piscicida in hybrid catfish is worrisome given current industry trends towards increased hybrid production. The project objectives were to assess intraspecific genetic variability of E. piscicida isolates recovered from diseased channel and hybrid catfish in Mississippi; and determine virulence associations among genetic variants. Repetitive extragenic palindromic sequence-based PCR (rep-PCR) using ERIC I and II primers was used to screen 158 E. piscicida diagnostic case isolates. A subsample of 39 E. piscicida isolates, representing predominant rep-PCR profiles, was further characterized using BOX and (GTG) 5 rep-PCR primers, virulence gene assessment and multilocus sequence analysis (MLSA) targeting housekeeping genes gyrb, pgi and phoU. The MLSA provided greater resolution than rep-PCR, revealing 5 discrete phylogroups that correlated similarly with virulence gene profiles. Virulence assessments using E. piscicida representatives from each MLSA group resulted in 14-day cumulative mortality ranging from 22% to 54% and 63 to 72% in channel and hybrid fingerlings, respectively. Across all phylogroups, mortality was higher in hybrid catfish (p < .05), supporting previous work indicating E. piscicida is an emerging threat to hybrid catfish aquaculture in the south-eastern United States., (© 2021 John Wiley & Sons Ltd.)

The recently discovered Tilapia parvovirus (TiPV) was the first Parvovirus confirmed to infect fish, causing mortality outbreaks in farmed adult Nile tilapia in China. Severe mortality outbreaks caused by Tilapia tilapinevirus (TiLV) to farmed tilapia in Thailand revealed the concomitant occurrence of TiPV. Out of ten fish farms screened, TiPV was detected in one site rearing juvenile red hybrid tilapia. Clinical signs included abnormal swimming, scale protrusion, skin and muscle haemorrhaging, exophthalmia and generalized anaemia. Histological findings showed extensive infiltration of lymphocytes, with increased melanomacrophage centres in the anterior kidney and spleen, erythrocyte depletion in the spleen and hepatic syncytial cells. Both TiLV and TiPV were systemically distributed in the body of moribund fish. The analysis of the near-complete TiPV genome isolated from Thailand revealed 98.74% sequence identity to the formerly isolated from China, together with a highly conserved and comparable genomic organization and with a 3 nucleotides deletion in the 5-UTR. The viral genome structure was highly conserved for each of its components, with nucleotide and amino acid identity ranging from 100% for ORF1 to 97% for ORF2, and with conserved HuH and Walker loop motifs within NS1. Taken together, our results document the first detection of TiPV outside China, thus for the first time in Thailand. Moreover, TiPV was detected for the first time during a natural occurrence in farmed red hybrid tilapia and involved in co-infection pattern with TiLV. Diagnostic investigations during tilapia disease outbreaks should include the screening for TiPV. Further studies are needed to elucidate TiPV genomic variance, pathobiology, including focussing on the outcomes of TiLV-TiPV co-infection patterns, necessary to enable risk assessment for the worldwide spreading of TiPV and to design adequate control measures against these emerging viruses in tilapia., (© 2021 Wiley-VCH GmbH.)

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD), causes significant economic losses worldwide, particularly in farmed Rainbow Trout Oncorhynchus mykiss. Over the last decade, multilocus sequence typing has revealed >30 clonal complexes (CCs) globally, comprised of >320 F. psychrophilum sequence types (STs). Despite the large number of CCs worldwide, CC-ST10, which is currently the largest CC affecting Rainbow Trout, has been the primary focus of F. psychrophilum virulence studies, leaving the role of other CCs as primary causes of BCWD epizootics unclear. To this end, fingerling Rainbow Trout were experimentally challenged with F. psychrophilum strains belonging to the CC now recognized as the second largest in the world (CC-ST191) alongside CC-ST10 strains. Cumulative percent mortality was 100% in 7-month-old Rainbow Trout and between 27.8% and 61.1% in 8-month-old Rainbow Trout. All examined F. psychrophilum STs were virulent to Rainbow Trout, and no significant differences in virulence between CC-ST10 and CC-ST191 were detected. Due to their wide distribution and high pathogenic potential, both CC-ST191 and CC-ST10 F. psychrophilum strains are excellent candidates for further research aimed at preventing and controlling BCWD., (© 2021 American Fisheries Society.)

Blue catfish alloherpesvirus (BCAHV) is a novel virus isolated from the blue catfish (Ictalurus furcatus). To date, the ultrastructure, virulence and immunogenicity of BCAHV have not been reported. Given the importance of blue catfish in producing channel ♀ (I. punctatus) × ♂ blue (I. furcatus) catfish hybrids and the increasing demand for hybrid catfish in the US catfish industry, the susceptibility of blue, channel and hybrid catfish to BCAHV was assessed. Further, the cross-protective potential of BCAHV against Ictalurid herpesvirus 1 (IcHV1) was investigated in channel and hybrid catfish that survive BCAHV exposure. Neutralization assays revealed BCAHV is refractive (neutralization index [NI] = 0) to anti-IcHV1 monoclonal antibody Mab 95, compared to IcHV1 (NI = 1.8). Exposure of blue catfish fingerling to 1.3 × 10 5 TCID 50 /L BCAHV produced cumulative mortality of 51.67 ± 0.70% and pathologic changes similar to those of channel catfish virus disease. No mortality was observed in channel or hybrid catfish. Twenty-eight days post-challenge, surviving channel and hybrid catfish were exposed to 9.4 × 10 4 TCID 50 /L IcHV1 (LC 50 dose), resulting in 100% relative per cent survival compared to naïve cohorts. These data provide baseline information for BCAHV and lay the groundwork for future studies. Data also identify BCAHV as a potential vaccine candidate against IcHV1. Based on host range and immunogenicity evaluations, in addition to genome sequence data from previous studies, BCAHV should be given consideration as a new species of Ictalurivirus., (© 2021 John Wiley & Sons Ltd.)

Edwardsiella piscicida is a Gram-negative facultative intracellular bacterium causing edwardsiellosis in catfish, the largest aquaculture industry in the United States. A safe and effective vaccine is an urgent need to avoid economic losses associated with E. piscicida outbreaks. PhoP/PhoQ is a two-component signal transduction system (TCS) that plays an important role in bacterial pathogenesis through sense and response to environmental and host stress signals. This study aimed to explore the contribution of PhoQ/PhoP in E. piscicida virulence and develop live attenuated vaccines against E. piscicida infection in channel catfish (Ictalurus punctatus) and hybrid catfish (channel catfish ♀ × blue catfish (I. furcatus) ♂). In the current study, two in-frame deletion mutants were constructed by deleting phoP (ETAC&#95;09785) and phoQ (ETAC&#95;09790) genes in E. piscicida strain C07-087, and the virulence and protection efficacy of the constructed strains were evaluated in catfish following intraperitoneal injection. Both EpΔphoP and EpΔphoQ strains had a delayed adaptation to oxidative stress (0.2% H 2 O 2 ) compared to E. piscicida wild type. The EpΔphoP and EpΔphoQ mutants produced significantly less biofilm compared to wild-type E. piscicida. Notably, EpΔphoP and EpΔphoQ mutants were significantly attenuated in channel catfish compared with wild-type E. piscicida (6.63% and 4.17% versus 49.16% mortalities), and channel catfish vaccinated with EpΔphoP and EpΔphoQ were significantly protected (95.65% and 97.92% survival) against E. piscicida infection at 21 days post-vaccination. In hybrid catfish, EpΔphoP was significantly more attenuated than EpΔphoQ, but EpΔphoQ provided significantly better protection than EpΔphoP. EpΔphoP and EpΔphoQ strains both induced specific antibodies in channel catfish against E. piscicida at 14 and 21 days post-vaccination. This result indicated that EpΔphoP and EpΔphoQ mutants were safe and protective in channel catfish fingerlings, while EpΔphoP was safe in hybrid catfish. Our findings show that PhoP and PhoQ are required for adaptation to oxidative stress and biofilm formation and may help E. piscicida face tough environmental challenges; thus, functional PhoP and PhoQ are critical for a successful infection., (© 2021 John Wiley & Sons Ltd.)

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully., (© 2021 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Immunoglobulins (Igs) are complex glycoproteins that play critical functions in innate and adaptive immunity of all jawed vertebrates. Given the unique characteristics of mucosal barriers, secretory Igs (sIgs) have specialized to maintain homeostasis and keep pathogens at bay at mucosal tissues from fish to mammals. In teleost fish, the three main IgH isotypes, IgM, IgD and IgT/Z can be found in different proportions at the mucosal secretions of the skin, gills, gut, nasal, buccal, and pharyngeal mucosae. Similar to the role of mammalian IgA, IgT plays a predominant role in fish mucosal immunity. Recent studies in IgT have illuminated the primordial role of sIgs in both microbiota homeostasis and pathogen control at mucosal sites. Ten years ago, IgT was discovered to be an immunoglobulin class specialized in mucosal immunity. Aiming at this 10-year anniversary, the goal of this review is to summarize the current status of the field of fish Igs since that discovery, while identifying knowledge gaps and future avenues that will move the field forward in both basic and applied science areas., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During 9-10 February 2018 and 21-22 February 2020, 7 adult Blue Suckers, Cycleptus elongatus, were collected by hoop nets from the Red River, Little River County (n = 3), and the Black River, Lawrence County (n = 4), Arkansas, and their gills, gallbladders, fins, integument, other major organs, and musculature were examined for myxozoans. All 7 (100%) were infected with an unknown species of gill-infecting Myxobolus sp. Twenty formalin-fixed plasmodia (cysts) of Myxobolus cloutmani n. sp. were elliptoidal, 407 μm long × 270 μm wide. Formalin-fixed myxospores were orbicular to broadly elliptoidal, 8.7 μm long × 7.8 μm wide. Two polar capsules were pyriform and subequal in size, extending over halfway in the myxospore. The larger polar capsule was 5.5 μm long × 3.1 μm wide, while the shorter was 5.1 × 2.9 μm. A coiled polar filament possessed 5 or 6 coils. The myxospore was 3.7 μm thick in sutural view, with a distinct sutural ridge. Qualitative and quantitative morphological data were from formalin-fixed as well as ethanol-preserved spores, while molecular data consisted of a 2,010 base pair sequence of the partial 18S ribosomal RNA gene and a 2,502 base pair sequence of the partial 28S ribosomal RNA gene. Phylogenetic analysis grouped M. cloutmani n. sp. with the other catostomid-infecting myxobolids. This is the first myxozoan reported from C. elongatus., (© American Society of Parasitologists 2021.)

Chinook Salmon Oncorhynchus tshawytscha is a keystone fish species in the Pacific Northwest. In 2019, unusual mortalities occurred in two different populations of cultured fingerlings from the same facility in California, USA. The systems consist of outdoor, enclosed, flow-through freshwater tanks that are maintained at 18 ± 1°C. Clinical signs and gross findings were only observed in one population and included abnormal swimming, inappetence, lethargy, skin discoloration, and the presence of multifocal nodular and ulcerative skin lesions. Microscopic lesions were infrequent and consisted of severe, locally extensive granulomatous dermatitis and myositis and mild, multifocal, granulomatous branchitis, myocarditis, and hepatitis. Intracellular acid-fast organisms were observed within areas of granulomatous myositis. Posterior kidney swabs were collected and inoculated in nutrient-rich and selective agar media and incubated at 25°C for 2 weeks. Visibly pure bacterial colonies were observed 7-10 d postinoculation. Partial sequences of 16S rRNA initially identified the recovered bacteria as members of the genus Mycobacterium. However, marked variability was observed among Mycobacterium spp. isolates by using repetitive extragenic palindromic polymerase chain reaction fingerprinting. Amplification and sequencing of the ribosomal RNA internal transcribed spacer, 65-kDa heat shock protein, and RNA polymerase β-subunit gene of the cultured isolates identified M. salmoniphilum and M. chelonae, discrete members of the M. chelonae-abscessus complex, isolated from diseased Chinook Salmon fingerlings., (© 2021 American Fisheries Society.)

Salmonid diseases caused by infections of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, remain difficult to manage as novel, pathogenic strains continue to emerge in aquaculture settings globally. To date, much of the research regarding treatment options and vaccine development has focused on rainbow trout (Oncorhynchus mykiss), but other inland-reared salmonids are also impacted by this Gram-negative bacterium. As such, Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis) were injection-challenged with a variety of previously reported F. psychrophilum strains isolated from disease diagnostic cases in salmonids, as well as a standard and well-studied F. psychrophilum strain (CSF 259-93) known to be virulent in rainbow trout. In three separate virulence assessments (Trials A, B and C), strains US063 (isolated from lake trout; Salvelinus namaycush) and US149 (isolated from Atlantic salmon) caused a significantly higher cumulative per cent mortality (CPM) relative to other strains in Atlantic salmon (p <.001 for all trials), with US149 causing significantly greater mortality than US063 in Trials A (CPM 97% vs. 65%, p =.008) and B (CPM 96% ± 2.3% vs. 81.33% ± 4.8%, p =.014). Trial C used a lower dose (1.86 × 10 8  CFU/mL) for US149, resulting in a lower mortality (78.67% ± 9.33%) relative to Trials A and B. CSF259-93 did not cause significant mortality in any trials. In brook trout, the strain 03-179 (originally isolated from steelhead trout; Oncorhynchus mykiss) was significantly more virulent than any other (CPM 100% ± 0%, p <.001), followed by US063 (73% ± 3.8%) and US149 (40% ± 6.1%,) respectively. Again, CSF259-93 did not cause significant mortality relative to a mock challenge treatment. Results provide information about the applicability of strain selection in F. psychrophilum virulence testing in Atlantic salmon and brook trout, demonstrating the high virulence of US063 and US149 for these salmonid species. This information is applicable for the development of therapeutics and vaccines against F. psychrophilum infections and demonstrates the reproducibility of the experimental challenge model., (© 2021 John Wiley & Sons Ltd.)