Enteric Septicemia Research Articles

Application of Loop-Mediated Isothermal Amplification for Edwardsiella Ictaluri Detection in Indonesia Enteric Septicemia of Catfish

Abstract Loop-mediated isothermal amplification (LAMP) method is useful for rapidly detecting Edwardsiella ictalurid infection, especially enteric septicemia of catfish (ESC). This study aims to investigate the LAMP method for E. ictaluri detection in ESC. This research was an experimental study using a total of 55 catfish, consisting of 5 negative controls and 50 catfish injected intraperitoneally with 0.1 mL of E. ictaluri at a concentration of 105 CFU/mL. The kidneys of three fishes were randomly selected 6, 12, 18, 24, 30, 36, 42, 48, and 54 hours after infection with E. ictaluri. In addition, samples were also collected on days 3, 5, and 7. Bacterial analysis was determined by conventional biochemistry (genus and species test), whole-genome sequencing of catfish, and LAMP amplification. All types of data obtained in this study were analyzed by descriptive statistics to compare infected and healthy catfish. The catfish were infected with E. ictaluri after 12 hours of infection according to the LAMP amplification procedure which revealed hemorrhages throughout the body, fins, protruding eyes, necrosis, inflammation of the spleen, liver, intestine, pale gills, gut, abdomen swelling, and tissue necrosis in the upper part of the head. It can be concluded that the LAMP method is more effective than the PCR method for detecting infection with E. ictaluri in catfish.

Expanding the Spectrum of Diseases and Disease Associations Caused by Edwardsiella tarda and Related Species

The genus Edwardsiella, previously residing in the family Enterobacteriaceae and now a member of the family Hafniaceae, is currently composed of five species, although the taxonomy of this genus is still unsettled. The genus can primarily be divided into two pathogenic groups: E. tarda strains are responsible for almost all human infections, and two other species (E. ictaluri, E. piscicida) cause diseases in fish. Human infections predominate in subtropical habitats of the world and in specific geospatial regions with gastrointestinal disease, bloodborne infections, and wound infections, the most common clinical presentations in decreasing order. Gastroenteritis can present in many different forms and mimic other intestinal disturbances. Chronic gastroenteritis is not uncommon. Septicemia is primarily found in persons with comorbid conditions including malignancies and liver disease. Mortality rates range from 9% to 28%. Most human infections are linked to one of several risk factors associated with freshwater or marine environments such as seafood consumption. In contrast, edwardsiellosis in fish is caused by two other species, in particular E. ictaluri. Both E. ictaluri and E. piscicida can cause massive outbreaks of disease in aquaculture systems worldwide, including enteric septicemia in channel catfish and tilapia. Collectively, these species are increasingly being recognized as important pathogens in clinical and veterinary medicine. This article highlights and provides a current perspective on the taxonomy, microbiology, epidemiology, and pathogenicity of this increasingly important group.

Edwarsiella ictaluri: Pathogenicity and LD50 in Pangasius nasutus

This study focuses on pathogenicity and LD50 of Pangasius nasutus against Edwardsiella ictaluri. Pangasius nasutus or ‘patin buah’ is a native freshwater species of peninsular Malaysia and can be found in Pahang River. The market price for this species is high as it tastes better compared to other catfish. Edwardsiella ictaluri is a pathogenic bacteria and the causative agent that cause enteric septicemia of catfish (ESC) in the Pangasius species. However, the prevention measures against Edwardsiella ictaluri are still unknown for Pangasius nasutus due to the lack of research and study. Therefore, the objective of this study is to identify Edwardsiella ictaluri isolated from Pangasius nasutus cultured in Pahang River using polymerase chain reaction (PCR) and to determine the pathogenicity and LD50 of Pangasius nasutus against Edwardsiella ictaluri through injection method. There are four different concentrations of Edwardsiella ictaluri (1×1010, 1×109, 1×108, 1×107) that were injected intraperitoneally including normal saline water as control to a total of 50 Pangasius nasutus. Observation of clinical signs and mortality were carried out for 30 days and LD50 was determined. The earliest clinical sign was observed at concentration of 1×1010 cfu/ml at 2 hours post-infection of Edwardsiella ictaluri where pale liver and congested kidney can be observed after dissection. Other clinical signs observed are inflammation on tail and fin, hemorrhagic fin, hemorrhagic upper mandible, discoloration, and inflammation on the lower part of body. The first mortality of Pangasius nasutus was at concentration 1×1010 cfu/ml at 2 hours of post infection. The highest cumulative mortality was recorded at concentration 1×1010 cfu/ml with 100% of mortality rate. From the result, the value of LD50 of Edwardsiella ictaluri calculated was 1×106 cfu/ml. From this study, it can be concluded that Edwardsiella ictaluri does affect the survivability Pangasius nasutus in Malaysia.

Trans-Cinnamaldehyde Primes More Robust Channel Catfish Immune Responses to Edwardsiella ictaluri Infection.

Infection with Edwardsiella ictaluri, a causative agent of enteric septicemia of catfish, threatens profitable catfish production through inventory losses. We previously demonstrated that trans-cinnamaldehyde (TC) enhances the survival of catfish following E. ictaluri infection. The present study was conducted to investigate catfish immune responses to TC feeding and E. ictaluri infection. The expression of 13 proinflammatory, innate, and adaptive immune-related genes was evaluated over time in two sets of experiments using real-time polymerase chain reaction (PCR). In the first experiment, catfish were fed a basal diet with or without TC supplementation, while in the second they were fed a TC-supplemented or normal diet followed by infection with E. ictaluri. The catfish group infected with E. ictaluri and fed a TC-diet showed significant changes in the expression of innate and adaptive immune-related genes compared to control group. At 21 and 28 days post-infection, recovered fish showed significant increases in the expression of IgM in the anterior kidney and spleen. These results suggest that the supplemental dietary intake of TC can improve the immune status of catfish via engaging innate and adaptive immune responses and the production of memory cells in immunocompetent tissues. Together, this study provides an important foundation for the potential application of TC as an antimicrobial alternative in aquaculture.

The RNA chaperone Hfq has a multifaceted role in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qβ replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of hfq in E. ictaluri remains unexplored. This study aimed to elucidate the role of hfq in E.ictaluri by creating an hfq mutant (EiΔhfq) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus Edwardsiella. The deletion of hfq resulted in a significantly reduced growth rate during the late exponential phase. Additionally, EiΔhfq displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, EiΔhfq demonstrated impaired growth, and we observed elevated hfq expression when subjected to in vitro and in vivo stress conditions. EiΔhfq exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that hfq is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the EiΔhfq vaccination completely protected catfish against subsequent EiWT infection. In summary, these results underscore the pivotal role of hfq in E. ictaluri, affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host.

Caracterización microbiológica y análisis genético de bacterias aisladas de hemocultivos y muestras fecales en terneros con síntomas de septicemia y diarrea

Diarrhea in calves can be caused by bacteria, viruses, and parasites. Among bacteria, Escherichia coli is considered responsible for the appearance of enteric diarrhea and septicemia in these animals, conditions that require immediate attention. Among E. coli infections of calves, more focus is placed on intestinal pathogenic (InPEC) infections, and extra – intestinal pathogenic (ExPEC) infections are ignored. This study aims to reveal which E. coli pathotype causes the infection as molecular and serotype and to reveal the differences according to the age groups of the factors in the herd. Blood and fecal samples of 10 calves aged 3 – 15 d with diarrhea were analyzed. The primary agent causing enteritis was determined by examining the stool samples with BoviD – 5 Ag. Then, samples were subjected to culture and identification processes. It was determined that the stool samples had 2/10 with E. coli K99, 4/10 with rotavirus, and 4/10 with mixed rotavirus infections and Cryptosporidium spp. E. coli was detected from all blood samples by hemoculture. The study isolated only the SepEC and ETEC groups from samples. All SepEC isolates were determined to carry type 1 pilus responsible for adhesion. In addition, it was determined that 9/10 of the SepEC group carried the colicin V gene responsible for pathogenicity. Also, all E. coli isolated from calves aged 3 – 15 d were found to be resistant to antibiotics. In conclusion, primary enteritis is caused by rotavirus Cryptosporidium spp. and ETEC. However, it was determined that SepEC group E. coli causing septicemia showed different antigenic and genetic features than E. coli in the intestinal tract. The virulence factors of the SepEC group may vary due to genomic plasticity, and their antigenic structures should be more closely examined and added to vaccine test studies.

Prevalence of Pangasius Fish Disease in Nepal

Objectives: This study was conducted to identify bacterial pathogens in both symptomatic and asymptomatic enteric septicemic infection of Pangasiaus hypophthalmus and evaluate their susceptibility to antibiotics. Methods: The totals of 120 kidney and liver swab samples of fish were collected from different places of Nepal. The samples were processed to identify the causative agent using pure culture of bacteria through streak plate techniques and different biochemical tests. To assess the susceptibility of the isolated bacteria to antibiotics Ciprofloxacin, Tetracycline, Doxycycline, Gentamycin, Ampicillin/ sulbactam, and Tobramycin were used. Results: The estimated prevalence of pathogenic bacteria is 27 per 10000 populations. Three pathogenic bacteria i.e. Edwardsiella ictaluri 57.7% (56/97), Aeromonas hydrophila 24.7% (24/97), and Pseudomonas fluorescence 17.5% (17/97) were identified in the studied asymptomatic and symptomatic Pangas fish disease. E. ictaluri was major isolates which was causative agent for the disease, enteric septicemia of catfish. Among the total bacterial isolates 100% bacterial isolates were sensitive to Ciprofloxacin and 84% to Gentamycin. Whereas 100% were resistance to Ampicillin and 84% to Tetracycline. And Tobramycin (60.8%) and Doxycycline (64.9%) showed intermediate result. Conclusion: Ciprofloxacin and Gentamycin could be administered to control enteric septicemia of catfish in Pangas. This study provides information to strategies to reduce economic losses to farmer and reduce possible health hazards.

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

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

Characterization of Type VI secretion system in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative facultative intracellular fish pathogen causing enteric septicemia of catfish (ESC). While various secretion systems contribute to E. ictaluri virulence, the Type VI secretion system (T6SS) remains poorly understood. In this study, we constructed 13 E. ictaluri T6SS mutants using splicing by overlap extension PCR and characterized them, assessing their uptake and survival in channel catfish (Ictalurus punctatus) peritoneal macrophages, attachment and invasion in channel catfish ovary (CCO) cells, in vitro stress resistance, and virulence and efficacy in channel catfish. Among the mutants, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO exhibited reduced replication inside peritoneal macrophages. EiΔevpM, EiΔevpN, and EiΔevpO showed significantly decreased attachment to CCO cells, while EiΔevpN and EiΔevpO also displayed reduced invasion of CCO cells (p < 0.05). Overall, T6SS mutants demonstrated enhanced resistance to oxidative and nitrosative stress in the nutrient-rich medium compared to the minimal medium. However, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO were susceptible to oxidative stress in both nutrient-rich and minimal medium. In fish challenges, EiΔevpD, EiΔevpE, EiΔevpG, EiΔevpJ, and EiΔevpK exhibited attenuation and provided effective protection against E. ictaluri wild-type (EiWT) infection in catfish fingerlings. However, their attenuation and protective efficacy were lower in catfish fry. These findings shed light on the role of the T6SS in E. ictaluri pathogenesis, highlighting its significance in intracellular survival, host cell attachment and invasion, stress resistance, and virulence. The attenuated T6SS mutants hold promise as potential candidates for protective immunization strategies in catfish fingerlings.

Isolation and screening of lactic acid bacteria producing anti-Edwardsiella from the gastrointestinal tract of wild catfish (Clarias gariepinus) for probiotic candidates

Abstract Introduction Members of lactic acid bacteria (LAB) have been well known for their antimicrobial activities against various bacterial pathogens in aquaculture species. Thus, the present study aimed at isolating LAB members from the intestinal tract of wild-caught catfish, Clarias gariepinus, and screening them for antimicrobial production against one of the most common bacterial pathogens, Edwardsiella ictaluri. Material and methods LAB were isolated from the intestinal tract of wild catfish caught at the Brantas River, East-Java Indonesia. Then, LAB were screened for antimicrobial activity against E. ictaluri by in vitro assays and further assessed for probiotic candidates. Results A total of 29 LAB were successfully isolated and further screened for anti-edwarsiella activities. Of the 29, six isolates had strong anti-edwardsiella activity (diameter of inhibition zone, &gt;10 mm). Based on their 16 s rRNA gene sequences, these LABs were identified as Lactococcus lactis, Enterococcus hirae, Weissella confusa, Weissella cibaria, and Enterococcus faecalis (two isolates). Further in vitro assays indicated that E. faecalis, L. lactis, W. confusa, and W. cibaria had good viability in the intestinal tract condition, having good adhesion capacity to intestinal mucus, and being harmless to catfish. However, two species (E. faecalis and W. cibaria) were resistant to novobiocin and enrofloxacin, respectively. Conclusion Three LAB species (E. faecalis, L. lactis, and W. confusa) are potential probiotic candidates in aquaculture to prevent enteric septicemia of catfish disease. However, further studies are needed to evaluate the use of probiotics in vivo.

Determination of Minimum Inhibitory Concentration (MIC) of Tigecycline against Salmonella typhi

Introduction: Typhoid fever, caused by S. Typhi, is prevalent in developing countries, particularly the Indian subcontinent. Salmonella infections can cause enteric fever, gastroenteritis, septicemia, and non-typhoidal Salmonellae (NTS) infections, especially in immunocompromised patients. Objective: To determine the Minimum Inhibitory Concentration (MIC) of Tigecycline against Salmonella Typhi. Methodology: This interventional study conducted at Department of Pharmacology &amp; Therapeutics in collaboration with Department of Microbiology at Ad-din Sakina Women’s Medical College, Jashore during March 2023 to April 2023.MIC of Tigecycline was determined by Broth Dilution Technique against standard strain of Salmonella typhi ATCC 24683. Result: The MIC of Tigecycline against Salmonella typhi was 2.0 μg/ml. Conclusion: Tigecycline is a potential therapeutic agent for Salmonella typhi infection, and should be restricted on the basis of blood culture and in MDR and XDR cases of typhoid fever only. The Journal of Ad-din Women's Medical College; Vol. 11 (2), July 2023; p 15-19

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.

Edwardsiella ictaluri Live Attenuated Vaccines Induce IgM Responses in Channel Catfish (Ictalurus punctatus)

Edwardsiella ictaluri (E. ictaluri), a Gram-negative intracellular pathogen, causes enteric septicemia of catfish (ESC). The primary role of B cells is to produce antibodies,

Edwardsiella ictaluri Almost Completely Occupies the Gut Microbiota of Fish Suffering from Enteric Septicemia of Catfish (Esc)

To reveal the changes in the gut microbiota of yellow catfish after being infected by Edwardsiella ictaluri, 16S rRNA gene high-throughput sequencing technology was used to analyze the microbial composition and diversity of the healthy and diseased yellow catfish. The gut microbial richness and diversity of the diseased fish were significantly lower than those of healthy fish. The composition and predicted function of yellow catfish gut microbiota were drastically altered after infection by E. ictaluri. Fusobacteriota, Proteobacteria, and Firmicutes were the predominant bacterial phyla in the gut of the healthy fish, while Proteobacteria was the dominant phylum in the gut of the diseased fish. At the genus level, the gut of healthy fish was dominated by Cetobacterium, Plesiomonas, and Romboutsia, while the gut of diseased fish was overwhelmed by the pathogenic E. ictaluri (99.22 ± 0.85%), and Cetobacterium, Plesiomonas, and Romboutsia disappeared. This is the most characteristic feature of the intestinal microbiota composition of yellow catfish edwardsiellosis. The same sequence of E. ictaluri was detected in the intestine of the healthy fish and the liver and intestine of the diseased fish. The anaerobic and Gram-positive bacteria were significantly decreased, and the digestive system, immune system, and metabolic functions of the gut microbiota were significantly reduced in the diseased fish gut. This may be part of the pathogenesis of fish edwardsiellosis.

Development of a polyclonal antibody Edwardsiella ictaluri serum for diagnosis of Enteric Septicemia of Catfish (ESC) in Patin Catfish (Pangasianodon hypopthalmus)

Enteric Septicemia of Catfish (ESC), caused by Edwarsiella ictaluri, has been considered a major bacterial disease in catfish, including Patin (Pangasionodon hypophthalmus), causing economic losses. Monitoring activities and identification of bacterial disease are performed using a series of biochemical tests, including Gram stain, Ryu, motility, and glucose; the test is at a certain temperature, requires specific conditions and facilities, and takes a long time. We developed a method based on antigen-antibody reactions to diagnose the disease, which provides more specific/accurate results in a shorter time to be used as a more flexible and rapid "diagnosis tool" as an anticipatory step in disease control. The development of antibody-serum is based on the specific character (specificity) of bacteria for antigen and performed according to standard methods using rabbits as test animals. The process starts with a mass culture of E. ictaluri, antigen in-activation, priming and booster injection, bleeding, and deriving polyclonal antibody serum. This diagnostic tool is expected to identify the target bacteria within 24 h, indicated by agglutination in the antigen-antibody reaction called the serum antibodies method. In this research, we produced serum, and ready to use, a 0.05-0.1 mL of serum-containing antibody is reacted with the bacterial isolate suspension.

Edwardsiella tarda- The Despised Threat to Nigerian Aquaculture and Human Health: A Review

Impediment to the aquaculture industry and consequently, impoverishment of the Nigerian populace can be attributed to several factors ranging from managemental to infectious or noninfectious. Despite the abundantly blessed land capacity and nourishment, the industry continues to face several losses. As part of the very important infectious agents known to cause high morbidity and mortality across several fish species is Edwardsiella tarda. E. tarda is directly associated with enteric septicemia in several fish species and ages. Infections caused by this bacterium is usually presented as gastrointestinal (gastroenteritis) and extra-intestinal (myonecrosis, bacteremia and septic arthritis) infections. E. tarda is an economic and public health threat because of its extensive impacts on the aquaculture industry as well as its ability to cause severe human infections such as diarrhea, gastroenteritis, typhoid-like illness, peritonitis with sepsis, cellulitis and meningitis. Furthermore, they are associated with the global antimicrobial resistance (AMR) in human and animal population. The threat posed by this bacterium have necessitated this current review on the overview of the risk factors and the presence of the organism in Nigeria. An elucidation into the bacterium, its epidemiology, threat to aquaculture industry and the global population are reviewed and the probable ways to control the presence of the organism are also discussed. Novel genetic engineering of the genes of the organism as a vital tool to drug and vaccine development are germane in this current age as a useful tool and strategy to have a world safe and in health. In conclusion, this review highlighted the necessary steps and approach to mitigate the bacterium and safe the Nigerian aquaculture from the emergence of a fatal infectious disease from fishes to human, as well as improvement in global trading.

Evaluation of the immune responses against enteric septicemia and determination of immune markers for selective breeding of pangasius families (Pangasianodon hypophthalmus)

The immune response of two groups of selective breeding panagasius families: susceptible (or sensitive) (S) and resistant (R) family groups, including red blood cell count (RBC count), white blood cell count (WBC count), neutrophil (NEU), monocyte (MONO), lymphocyte (LYM), and melano-macrophage centers (MMCs) count in liver, kidney and spleen tissue and antibody titers (ABT) against the bacteria Edwardsiella ictaluriwere evaluated in this research. Fish were sampled at five truncated points after infection with the bacteria Edwardsiella ictaluri including before the challenge, 24, 48, 264, and 312 hpi. The immune response of the R group was more effective than that of the S group, showing: (1) RBC decreased less during the challenge; (2) WBC, NEU, MONO, LYM increased more rapidly, especially at the truncated point 24-48 hpi; (3) MMCs in liver, kidney, and spleen of the R group increased faster up to the truncated point 48 hpi while the S group increased more slowly and only up to the truncated point 24 hpi; (4) ABT were higher in all stages of the challenge. The research developed a multivariable logistic regression model with immune markers such as NEU, ABT and MMCs in the liver at the truncated point of 24-48 hpi to identify individuals which were resistant to Enteric Septicemia of Catfish (ESC), with sensitivity and specificity of 91.7, 95.0%, respectively.

Pathological and Ultrastructural Characterization of an Edwardsiella ictaluri Triple hemR Mutant

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.

Development of a hyper-adhesive and attenuated Edwardsiella ictaluri strain as a novel immersion vaccine candidate in yellow catfish (Pelteobagrus fulvidraco)

Edwardsiella ictaluri, a Gram-negative intracellular pathogen, is the causative agent of enteric septicemia in channel catfish, and catfish aquaculture in China suffers heavy economic losses due to E. ictaluri infection. Vaccination is an effective control measure for this disease. In this study, an attenuated E. ictaluri strain was acquired through deletion mutation of the T3SS protein eseJei, and the ΔeseJei strain fails to replicate in the epithelioma papillosum of carp cells. The type 1 fimbria plays a pivotal role in the adhesion of E. ictaluri, and it was found in this study that deletion of −245 to −50 nt upstream of fimA increases its adhesion to around five times that of the WT strain. A hyper-adhesive and highly attenuated double mutant (ΔeseJeiΔfimA-245--50 strain) was constructed, and it was used as a vaccine candidate in yellow catfish via bath immersion at a dosage of 1 × 105 CFU/mL. It was found that this vaccine candidate can stimulate protection when challenged with E. ictaluri HSN-1 at 5 × 107 CFU/mL (∼20 × LD50). The survival rate was 83.61% for the vaccinated group and 33.33% for the sham-vaccinated group. The RPS (relative percent of survival) of the vaccination trial reached 75.41%. In conclusion, the ΔeseJeiΔfimA-245--50 strain developed in this study can be used as a vaccine candidate. It excels in terms of ease of delivery (via bath immersion) and is highly efficient in stimulating protection against E. ictaluri infection.

CRISPR/Cas-9 induced knockout of myostatin gene improves growth and disease resistance in channel catfish (Ictalurus punctatus)

Channel catfish, Ictalurus punctatus, is the most produced aquaculture species in the United States, although production levels have decreased since their peak in 2003. Myostatin (mstn) regulates skeletal muscle growth and has been identified as an important gene for increasing body weight in aquaculture species. In this study, the effects of CRISPR/Cas9 knockout of the mstn gene in channel catfish were investigated. Three sgRNAs targeting exon 1 of the channel catfish myostatin gene were microinjected with Cas9 protein in embryos. A total of 209 fish survived to 6 mph after microinjection of Cas9 and sgRNA targeting exon 1 of the mstn gene over the 3 yr experiment (2017–2019) with an average mutation rate of 58%. Successful generation of myostatin knockout (MSTN KO) F1 heterozygotes was achieved in 2019 by individually mating two pairs of control females with MSTN homozygous KO males. The offspring of both families inherited the mutation at a mean rate of 88%. Growth was generally higher in MSTN mutants than controls at all life stages and environments. P1 MSTN mutants were 88% larger than controls at the stocker stage (100 to 200 g) and 27% larger than controls at market size. Heterozygous F1 mutants were 218% larger than controls at the stocker stage. MSTN mutants had reduced overall expression levels of mstn compared to controls. When challenged with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish, MSTN mutants performed equally or better than controls. With a high mutation rate and inheritance as well as improved growth and disease resistance, using MSTN gene-edited channel catfish could greatly benefit commercial farms.