Assessment of dietary yeast-based additives for cultured catfish and tilapia health.

Channel catfish (Ictalurus punctatus) and Nile tilapia (Oreochromis niloticus) are two aquaculture species of great importance. Intensive production is often hindered by poor growth performance and disease mortality. The aim of this study was to evaluate the potential of a commercial fermented yeast product, DVAQUA, on channel catfish and Nile tilapia growth performance metrics and disease resistance. Channel catfish and Nile tilapia were fed practical diets supplemented with 0%, 0.1% or 0.4% of DVAQUA over approximately 2-month feeding periods in recirculation aquaculture systems. To assess the potential of the postbiotic against common aquaculture pathogens, juvenile catfish were subsequently challenged by immersion with Edwardsiella ictaluri S97-773 or virulent Aeromonas hydrophila ML09-119. Nile tilapia juveniles were challenged by injection with Streptococcus iniae ARS-98-60. Serum lysozyme activity, blood chemistry and growth metrics were measured at the end of the feeding period, but no differences were observed across the different metrics, except for survival. For the pathogen challenges, there were no differences in endpoint mortality for channel catfish with either pathogen (p > .05). In contrast, Nile tilapia survivability to S. iniae infection increased proportionally to the inclusion of DVAQUA (p = .005). Changes to sera lysozyme activity were also noted in the tilapia trial, with a reduction of activity in the fish fed the 0.4% DVAQUA diet compared to the control diet (p = .031). Expression profiles of proinflammatory genes and antibodies were also found to be modulated in channel catfish fed the postbiotic, indicating some degree of protective response. These results suggest that this postbiotic may be beneficial in protecting Nile tilapia against S. iniae infection by influencing immune parameters and additional research is needed to evaluate the potential of this DVAQUA for improving catfish health and disease control.

Practical diets for California yellowtail, Seriola dorsalis: Use of advanced soybean meal products on growth performance, body composition, intestinal morphology, and immune gene expression.

California yellowtail (CYT), Seriola dorsalis, is a promising candidate for aquaculture due to its rapid growth and high-quality flesh, particularly in markets like Japan, Australia, China, and the United States. Soy protein has shown success as a replacement for marine protein sources in CYT diets, reducing fishmeal levels, though concerns about potential intestinal inflammation persist with the inclusion of solvent-extracted soybean meal. To address this, processing strategies like fractionation, enzymatic treatment, heat treatment, and microbial fermentation have been employed to mitigate the negative impacts of soybean meal on fish nutrition and immune systems. This study focuses on optimizing soybean meal inclusion levels by incorporating advanced soy variants into CYT diets. The eight-week feeding trial, conducted in a recirculation system, featured six diets with sequential inclusion levels (0, 50, 100%) of high protein low oligosaccharide soybean meal (Bright Day, Benson Hill, St Louis, MO) and enzyme-treated soybean meal (HP 300, Hamlet Protein Inc., Findlay, OH), replacing solvent-extracted soybean. The study compares these formulations against a soy-free animal protein-based diet. At the end of the trial, fish were sampled for growth performance, body proximate composition, intestinal morphology, and immune response from gut samples. Results showed consistent FCR (P = 0.775), weight gain (P = 0.242), and high survival rate (99.4 ± 0.5%) among dietary treatments (P>0.05). Histological evaluations revealed no gut inflammation and gene expression analysis demonstrated no significant variations in immune, physiological, and digestive markers apn (P = 0.687), mga (P = 0.397), gpx1 (P = 0.279), atpase (P = 0.590), il1ß (P = 0.659). The study concludes that incorporating advanced soybean meal products, replacing up to 20% of fishmeal does not negatively affect CYT's growth and intestinal health. This suggests that all three soy sources, contributing 35% of total protein (15.4 g 100 g-1 diet), can be included in practical diets without compromising CYT's intestinal integrity or growth. These findings have positive implications for the commercial production of CYT and future research on the incorporation of plant-based proteins in aquaculture diets.

From heterosis to outbreeding depression: genotype-by-environment interaction shifts hybrid fitness in opposite directions.

In F1 hybrids, phenotypic values are expected to be near the parental means under additive effects or close to one parent under dominance. However, F1 traits can fall outside the parental range, and outbreeding depression occurs when inferior fitness is observed in hybrids. Another possible outcome is heterosis, a phenomenon that interspecific hybrids or intraspecific crossbred F1s exhibit improved fitness compared to both parental species or strains. As an application of heterosis, hybrids between channel catfish females and blue catfish males are superior in feed conversion efficiency, carcass yield, and harvestability. Over 20 years of hybrid catfish production in experimental settings and farming practices generated abundant phenotypic data, making it an ideal system to investigate heterosis. In this study, we characterized fitness in terms of growth and survival longitudinally, revealing environment-dependent heterosis. In ponds, hybrids outgrow both parents due to an extra rapid growth phase of 2-4 months in year 2. This bimodal growth pattern is unique to F1 hybrids in pond culture environments only. In sharp contrast, the same genetic types cultured in tanks display outbreeding depression, where hybrids perform poorly, while channel catfish demonstrate superiority in growth throughout development. Our findings represent the first example, known to the authors, of opposite fitness shifts in response to environmental changes in interspecific vertebrate hybrids, suggesting a broader fitness landscape for F1 hybrids. Future genomic studies based on this experiment will help understand genome-environment interaction in shaping the F1 progeny fitness in the scenario of environment-dependent heterosis and outbreeding depression.

Transcriptome analysis revealed potential mechanisms of channel catfish growth advantage over blue catfish in a tank culture environment.

Channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus) are two economically important freshwater aquaculture species in the United States, with channel catfish contributing to nearly half of the country's aquaculture production. While differences in economic traits such as growth rate and disease resistance have been noted, the extent of transcriptomic variance across various tissues between these species remains largely unexplored. The hybridization of female channel catfish with male blue catfish has led to the development of superior hybrid catfish breeds that exhibit enhanced growth rates and improved disease resistance, which dominate more than half of the total US catfish production. While hybrid catfish have significant growth advantages in earthen ponds, channel catfish were reported to grow faster in tank culture environments. In this study, we confirmed channel fish's superiority in growth over blue catfish in 60-L tanks at 10.8 months of age (30.3 g and 11.6 g in this study, respectively; p < 0.001). In addition, we conducted RNA sequencing experiments and established transcriptomic resources for the heart, liver, intestine, mucus, and muscle of both species. The number of expressed genes varied across tissues, ranging from 5,036 in the muscle to over 20,000 in the mucus. Gene Ontology analysis has revealed the functional specificity of differentially expressed genes within their respective tissues, with significant pathway enrichment in metabolic pathways, immune activity, and stress responses. Noteworthy tissue-specific marker genes, including lrrc10, fabp2, myog, pth1a, hspa9, cyp21a2, agt, and ngtb, have been identified. This transcriptome resource is poised to support future investigations into the molecular mechanisms underlying environment-dependent heterosis and advance genetic breeding efforts of hybrid catfish.

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

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

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

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

Investigation of dietary exogenous protease and humic substance on growth, disease resistance to Flavobacterium covae and immune responses in juvenile channel catfish (Ictalurus punctatus).

Many bacterial pathogens impact the US catfish industry, and disease control can be challenging for producers. Columnaris disease in channel catfish, Ictalurus punctatus, is primarily caused by Flavobacterium covae (formerly F. columnare). Immunostimulants may enhance nonspecific immune responses and offer an alternative to antibiotic treatments in catfish. Furthermore, dietary protein sources and inclusions are also essential to fish health and nutrition and may enhance overall fish performance in pond culture. The current project evaluated two immunostimulants: a protease complex (PC) and a humic substance (HS) derived from a reed-sedge peat product. A 60-day trial examined the effects of five diets on growth performance, immune response and resistance to experimental F. covae infection in channel catfish. Diets included a high-quality fishmeal diet (32%; CF32), a high-protein soy-based diet (32%; C32), a low-protein soy-based diet (28%; C28; predominately used in industry), a low-protein soy diet supplemented with C28 + PC at 175 g metric ton-1 and C28 + HS in a low-protein diet at 23 g metric ton-1 . Following feeding for 60 d, juvenile channel catfish were sampled for growth performance and baseline health indicators (n = 3; body mucus, blood for sera, kidney and spleen). A subset of fish was then subjected to an immersion-based in vivo challenge trial with F. covae (ALG-00-530; 106 CFU mL-1 exposure). At 60d post-initiation, there were no dietary differences in the relative growth rate (p = .063) or thermal growth coefficient (p = .055), but the 32% diets generally appeared to perform best. Post-challenge, the C32 group's mortality was higher than the C28 + PC (p = .006) and C28 + HS diets (p = .005). Although not significant, the C28 and CF32 groups also demonstrated higher mortality compared to both PC and HS diets. Sera lysozyme concentration was found to increase following pathogen challenge (p < .001) and in comparison with mock-challenged catfish (p < .001). Elevated expression levels of proinflammatory cytokines (il-1ß, il-8, tnf-α and tgf-ß) were observed at trial midpoint and post-infection when compared to 60d. The C28 treatment was found to have lower tnf-α expression than the C28 + PC (p = .042) and C28 + HS (p = .042) groups following exposure to F. covae. These challenge data suggest that the immunostimulants (PC and HS) in plant-based protein may be beneficial in protecting against F. covae when offered in low-protein channel catfish diets.

Probiotics enhance resistance to Streptococcus iniae in Nile tilapia (Oreochromis niloticus) reared in biofloc systems.

Biofloc technology is a rearing technique that maintains desired water quality by manipulating carbon and nitrogen and their inherent mixture of organic matter and microbes. Beneficial microorganisms in biofloc systems produce bioactive metabolites that may deter the growth of pathogenic microbes. As little is known about the interaction of biofloc systems and the addition of probiotics, this study focused on this integration to manipulate the microbial community and its interactions within biofloc systems. The present study evaluated two probiotics (B. velezensis AP193 and BiOWiSH FeedBuilder Syn 3) for use in Nile tilapia (Oreochromis niloticus) culture in a biofloc system. Nine independent 3785 L circular tanks were stocked with 120 juveniles (71.4 ± 4.4 g). Tilapia were fed for 16 weeks and randomly assigned three diets: a commercial control diet or a commercial diet top-coated with either AP193 or BiOWiSH FeedBuilder Syn3. At 14 weeks, the fish were challenged with a low dose of Streptococcus iniae (ARS-98-60, 7.2 × 107 CFU mL-1 , via intraperitoneal injection) in a common garden experimental design. At 16 weeks, the fish were challenged with a high dose of S. iniae (6.6 × 108 CFU mL-1 ) in the same manner. At the end of each challenge trial, cumulative per cent mortality, lysozyme activity and expression of 4 genes (il-1ß, il6, il8 and tnfα) from the spleen were measured. In both challenges, the mortalities of the probiotic-fed groups were significantly lower (p < .05) than in the control diet. Although there were some strong trends, probiotic applications did not result in significant immune gene expression changes related to diet during the pre-trial period and following exposure to S. iniae. Nonetheless, overall il6 expression was lower in fish challenged with a high dose of ARS-98-60, while tnfα expression was lower in fish subjected to a lower pathogen dose. Study findings demonstrate the applicability of probiotics as a dietary supplement for tilapia reared in biofloc systems.

Investigating the Ability of Edwardsiella ictaluri and Flavobacterium covae to Persist within Commercial Catfish Pond Sediments under Laboratory Conditions.

Two prevalent bacterial diseases in catfish aquaculture are enteric septicemia of catfish and columnaris disease caused by Edwardsiella ictaluri and Flavobacterium covae, respectively. Chronic and recurring outbreaks of these bacterial pathogens result in significant economic losses for producers annually. Determining if these pathogens can persist within sediments of commercial ponds is paramount. Experimental persistence trials (PT) were conducted to evaluate the persistence of E. ictaluri and F. covae in pond sediments. Twelve test chambers containing 120 g of sterilized sediment from four commercial catfish ponds were inoculated with either E. ictaluri (S97-773) or F. covae (ALG-00-530) and filled with 8 L of disinfected water. At 1, 2, 4-, 6-, 8-, and 15-days post-inoculation, 1 g of sediment was removed, and colony-forming units (CFU) were enumerated on selective media using 6 × 6 drop plate methods. E. ictaluri population peaked on Day 3 at 6.4 ± 0.5 log10 CFU g-1. Correlation analysis revealed no correlation between the sediment physicochemical parameters and E. ictaluri log10 CFU g-1. However, no viable F. covae colonies were recovered after two PT attempts. Future studies to improve understanding of E. ictaluri pathogenesis and persistence, and potential F. covae persistence in pond bottom sediments are needed.

Characterization of maternal immunity following vaccination of broodstock against IHNV or Flavobacterium psychrophilum in rainbow trout (Oncorhynchusmykiss).

Infectious hematopoietic necrosis (IHN) is a significant viral disease affecting salmonids, whereas Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD), remains one of the most significant bacterial pathogens of salmonids. We explored maternal immunity in the context of IHN and BCWD management in rainbow trout (Oncorhynchus mykiss) aquaculture. Two experimental trials were conducted where different groups of female broodstock were immunized prior to spawning with an IHNV DNA vaccine or a live attenuated F. psychrophilum (Fp B.17-ILM) vaccine alone, or in combination. Progeny were challenged with either a low or high dose of IHNV at 13 days post hatch (dph) and 32 dph or challenged with F. psychrophilum at 13 dph. Mortality following a low-dose IHNV challenge at 13 dph was significantly lower in progeny from vaccinated broodstock vs. unvaccinated broodstock, but no significant differences were observed at 32 dph. Mortality due to BCWD was also significantly reduced in 13 dph fry that originated from broodstock immunized with the Fp B.17-ILM vaccine. After vaccination broodstock developed specific or neutralizing antibodies respectively to F. psychrophilum and IHNV; however, antibody titers in eggs and fry were undetectable. In the eggs and fry mRNA transcripts of the complement components C3 and C5 were detected at much higher levels in progeny from vaccinated broodstock and showed a significantly increased and rapid response post-challenge compared with unvaccinated broodstock. After challenges pro-inflammatory cytokine expression was immediately and considerably elevated in the fry from vaccinated broodstock vs. unvaccinated broodstock, whereas adaptive immune genes were elevated to a lesser degree. Results suggest that maternal transfer of innate and adaptive factors at the transcript level occurred because development of lymphomyeloid organs is not complete in such young fry. In addition to documenting maternally derived immunity in teleosts, this study demonstrates that broodstock vaccination can confer some degree of protection to progeny against viral and bacterial pathogens.

Development of a monoclonal antibody specific to burbot (Lota lota) IgM and optimization of an ELISA to measure anti-Aeromonas sp. antibody titers following pathogen challenge.

Burbot (Lota lota) are an ideal candidate for cool or cold-water aquaculture and are gaining interest because of their high economic value, low temperature requirements, and fast growth rate. Limited information exists on the innate and adaptive immune systems of this species. This is partly due to the lack of species-specific tools to determine antibody responses following disease or vaccination or to characterize the immune response in general. An anti-IgM monoclonal antibody (mAb 27C) was developed and characterized via enzyme-linked immunosorbent assay (ELISA) and Western blot for species specificity, affinity to the heavy chain of burbot IgM, and cross-reactivity to other reagents used in the analysis. The 27C monoclonal antibody was further utilized to develop an ELISA protocol to measure the specific antibody response of burbot following exposure to two pathogenic strains of Aeromonas sp. (A141 and IR004). This ELISA confirmed that vaccinated burbot that survived the challenge with either strain developed statistically higher titers of anti-Aeromonas antibodies specific for the relative strain when compared to fish that were not vaccinated or challenged. Western blot analysis further demonstrated that burbot surviving challenge had serum IgM that recognized distinct antigens specific to the strain they were challenged with, A141 bound to antigens in the 50-250Kda range and IR004 bound to a distinct 150Kda antigen. Western blots further indicated that each strain shared antigenic regions regardless of experimental Aeromonas strain exposure. Finally, immunofluorescent staining confirmed that mAb 27C binds to membrane-bound IgM (presumably B cells) on burbot head kidney cells. Taken together, results from this study demonstrate that mAb 27C specifically recognized burbot IgM and will be an important tool to further characterize the adaptive and cellular immune responses of this fish species.

A recombinant 9E1 monoclonal antibody binds membrane and soluble channel catfish immunoglobulin M.

The development and validation of the recombinant 9E1 monoclonal antibody against channel catfish IgM is described. The variable heavy and light chain domains of the 9E1 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into 293F cells and mature IgG was purified from culture supernatant. It is demonstrated that the recombinant 9E1 monoclonal antibody binds to soluble IgM in ELISA and ELISPOT assays and to membrane-bound IgM by immunofluorescence with different B-cell types. The recombinant 9E1 monoclonal antibody will be a valuable tool in the continued examination of the channel catfish adaptive immune system.

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

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

Persistence of a Wild-Type Virulent Aeromonas hydrophila Isolate in Pond Sediments from Commercial Catfish Ponds: A Laboratory Study.

Virulent Aeromonas hydrophila (vAh) is a major bacterial pathogen in the U.S. catfish industry and is responsible for large-scale losses within commercial ponds. Administering antibiotic feeds can effectively treat vAh infections, but it is imperative to discern new approaches and better understand the mechanics of infection for this bacterium. As such, the persistence of vAh in pond sediments was determined by conducting laboratory trials using sediment from four commercial catfish ponds. Twelve chambers contained sterilized sediment, vAh isolate ML-09-119, and 8 L of water maintained at 28 °C and were aerated daily. At 1, 2, 4, 6, and 8 days, and every 7th day post-inoculation for 28 days, 1 g of sediment was removed, and vAh colony forming units (CFU) were enumerated on ampicillin dextrin agar. Viable vAh colonies were present in all sediments at all sampling periods. The vAh growth curve peaked (1.33 ± 0.26 × 109 CFU g-1) at 96 h post-inoculation. The population plateaued between days 14 and 28. No correlations were found between CFU g-1 and physiochemical sediment variables. This study validated the ability of vAh to persist within pond sediments in a laboratory setting. Further research on environmental factors influencing vAh survivability and population dynamics in ponds is needed.

Comparison of injection and immersion challenges of Renibacterium salmoninarum strains in Rainbow Trout.

OBJECTIVE: Renibacterium salmoninarum is a pathogenic gram-positive bacterium and is the causative agent of bacterial kidney disease (BKD), a malady that mainly impacts salmonid species. Experimental challenges were conducted to assess the virulence and challenge route for select R. salmoninarum strains (CK-90 and ATCC 33739) in Rainbow Trout Oncorhynchus mykiss. METHODS: The CK-90 strain was intracoelomically injected (100 µL) at a high dose containing 4.80 × 106 CFU/g of fish (optical density at 525 nm [OD525 ] = 1.779) and a low dose containing 6.86 × 105 CFU/g of fish (OD525  = 1.077); alternatively, fish were immersed in a solution containing 4.5 × 107 CFU/mL of fish (OD525  = 0.886). The ATCC 33739 strain (originating from Brook Trout Salvelinus fontinalis) was also included and intracoelomically injected at 3.58 × 105 CFU/g of fish (OD525  = 1.431) to discern differences in virulence between the strains. RESULT: Clinical signs of BKD manifested at approximately 10 d postchallenge, and mortalities began at 19 days postchallenge. To confirm infection and quantify R. salmoninarum antigen load, an enzyme-linked immunosorbent assay (ELISA) was conducted using kidney tissue collected after the challenge. Rainbow Trout that were challenged with CK-90 by injection (both high- and low-dose groups) exhibited significantly higher mortality than fish that were injected with ATCC 33739 or those that were exposed to CK-90 via immersion challenge. The R. salmoninarum p57 (57-kDa protein) antigen was confirmed via ELISA. Antigen load for fish injected with CK-90 (high dose: OD405  = 0.71; low dose: OD405  = 0.66) was significantly higher than that for fish injected with ATCC 33739 (OD405  = 0.34). The CK-90 strain (both high and low doses) was more virulent than ATCC 33739, which caused no mortalities over the 28-days trial. Although there were no mortalities among ATCC 33739 fish, the ELISA confirmed that the R. salmoninarum antigen infiltrated kidney tissue in those fish. CONCLUSION: The immersion challenge methodology for R. salmoninarum CK-90 was ineffective for inducing mortalities at the examined dose.

Growth Performance, Survival, Blood Chemistry, and Immune Gene Expression of Channel Catfish (Ictalurus punctatus) Fed Probiotic-Supplemented Diets.

The channel catfish (Ictalurus punctatus) farming industry is the largest and one of the oldest aquaculture industries in the United States. Despite being an established industry, production issues stemming from disease outbreaks remain problematic for producers. Supplementing fish diets with probiotics to enhance the immune system and growth potential is one approach to mitigating disease. Although considerable laboratory data demonstrate efficacy, these results do not always translate to natural modes of disease transmission. Hence, the present work was conducted in the laboratory but incorporated flow-through water from large catfish pond production systems, allowing for natural exposure to pathogens. Two feeding trials were conducted in an 18-tank aquaria system housing two different sizes, 34.8 ± 12.5 g and 0.36 ± 0.03 g, of channel catfish. Channel catfish in the first trial were fed three experimental diets over six weeks. Commercial diets were top-coated with two selected spore-forming Bacillus spp. probiotics, Bacillus velezensis AP193 (1 × 106 CFU g−1) and BiOWiSH (3.6 × 104 CFU g−1), or a basal diet that contained no dietary additive. In the second eight-week trial, diets were top-coated with BiOWiSH at three concentrations (1.8, 3.6, and 7.3 × 104 CFU g−1), along with one basal diet (no probiotic). At the completion of these studies, growth performance, survival, hematocrit, blood chemistry, and immune expression of interleukin 1ß (il1ß), tumor necrosis factor-alpha (tnf-α), interleukin-8 (il8), transforming-growth factor ß1 (tgf-ß1), and toll-like receptor 9 (tlr9) were evaluated using qPCR. Trial results revealed no differences (p > 0.05) among treatments concerning growth, survival, or hematological parameters. For immune gene expression, interesting trends were discerned, with substantial downregulation observed in B. velezensis AP193-fed fish for il1ß, tnf-α, and tlr9 expression within splenic tissue, compared to that of the basal and BiOWiSH diets (p < 0.05). However, the results were not statistically significant for anterior kidney tissue in the first trial. In the second trial, varied levels of probiotic inclusion revealed no significant impact of BiOWiSH's products on the expression of il1ß, tnf-α, il8, and tgf-ß1 in both spleen and kidney tissue at any rate of probiotic inclusion (p > 0.05). Based on these findings, more research on utilizing probiotics in flow-through systems with natural infection conditions is crucial to ensure consistency from a controlled laboratory scale to real-world practices.

Multi-tissue RNAseq reveals genetic and temporal differences in acute response to viral (IHNV) infection among three selected lines of rainbow trout with varying resistance.

Utilizing RNA-seq, this study compared the transcriptomic responses of three improved strains (VSel, PSel, and CSel) of rainbow trout fry during acute stages of challenge with infectious hematopoietic necrosis virus (IHNV). The VSel strain has been selected for resistance against the specific strain of IHNV used in our challenge, PSel has undergone selection for utilization of plant-protein based feeds and previously has shown elevated non-specific disease resistance despite no disease related selection pressures, and the final strain, CSel, is a commercial strain that has been domesticated for several years but has not been selected for specific viral disease resistance. Following a 21-day IHNV challenge, Kaplan-Meier survival estimator curves and cumulative percent mortality (CPM) showed significant differences in IHNV resistance across strains: VSel - 19.3 ± 5.0%, PSel - 67. ± 3.03%, CSel - 94.6 ± 4.1% CPM. To evaluate acute responses to IHNV infection, whole blood, as well as samples from the kidney, liver, and intestine, were collected at 0, 4, 12, 24, and 48 h post infection (hpi). Serum lysozyme activity, a marker of non-specific innate immunity, showed strain and temporal effects during the acute infection phase with PSel showing the highest activity at 0 and 48 hpi. Differential gene expression responses were detected, with varying degrees, in all tissues, both between strains, as well as across acute timepoints within strains. The VSel strain showed upregulation for a particular subset of viral recognition genes during early infection timepoints and rather limited upregulation of immune genes later, while maintaining and reactivating metabolic pathways. The CSel strain showed a downregulation of metabolic related genes and a limited upregulation of immune genes, while the PSel strain showed similar downregulation of metabolic genes during acute infection, yet when compared to the CSel strain, showed a more robust innate immune response. Evaluation of upregulated immune response genes, as well as interferon-related genes showed the PSel strain to have the greatest number of uniquely upregulated immune genes in both the kidney and intestine, with CSel and PSel showing a similar number of such genes upregulated in liver. A moderate number of immune response genes were shared between PSel and CSel in all tissues, though both PSel and VSel showed a high number of uniquely overexpressed immune response genes in the kidney, and PSel showed the highest number of uniquely upregulated interferon related genes in the intestine. Overall, the VSel response was unique from the CSel with very little overlap in activated immune responses. Findings from this study highlight the disparity in IHNV resistance among genetic strains of rainbow trout, while identifying molecular mechanisms underlying differences in disease phenotypes. Furthermore, our results on trout strains with distinct selection backgrounds yields comparative insights into the adaptive gains brought about by selection programs for pathogen-specific disease resistance, as well as the non-specific immune enhancement associated with selection for utilization of plant-based diets.

Environment-Dependent Heterosis and Transgressive Gene Expression in Reciprocal Hybrids between the Channel Catfish Ictalurus punctatus and the Blue Catfish Ictalurus furcatus.

The hybrid between female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) is superior in feed conversion, disease resistance, carcass yield, and harvestability compared to both parental species. However, heterosis and heterobeltiosis only occur in pond culture, and channel catfish grow much faster than the other genetic types in small culture units. This environment-dependent heterosis is intriguing, but the underlying genetic mechanisms are not well understood. In this study, phenotypic characterization and transcriptomic analyses were performed in the channel catfish, blue catfish, and their reciprocal F1s reared in tanks. The results showed that the channel catfish is superior in growth-related morphometrics, presumably due to significantly lower innate immune function, as investigated by reduced lysozyme activity and alternative complement activity. RNA-seq analysis revealed that genes involved in fatty acid metabolism/transport are significantly upregulated in channel catfish compared to blue catfish and hybrids, which also contributes to the growth phenotype. Interestingly, hybrids have a 40-80% elevation in blood glucose than the parental species, which can be explained by a phenomenon called transgressive expression (overexpression/underexpression in F1s than the parental species). A total of 1140 transgressive genes were identified in F1 hybrids, indicating that 8.5% of the transcriptome displayed transgressive expression. Transgressive genes upregulated in F1s are enriched for glycan degradation function, directly related to the increase in blood glucose level. This study is the first to explore molecular mechanisms of environment-dependent heterosis/heterobeltiosis in a vertebrate species and sheds light on the regulation and evolution of heterosis vs. hybrid incompatibility.