Virulent Aeromonas Hydrophila Research Articles

Innate and adaptive immunity gene expression profiles induced by virulent Aeromonas hydrophila infection in the immune-related organs of channel catfish

Aeromonas hydrophila causes motile Aeromonas septicemia (MAS) in freshwater fish. In recent years, MAS outbreaks due to virulent Aeromonas hydrophila (vAh) have been responsible for large-scale losses within commercial catfish farms in Mississippi and Alabama. The aim of this study was to evaluate immune gene expression in catfish immune-competent tissues during infection with vAh strain ML09-119. Specific pathogen-free catfish fingerlings were intraperitoneally infected with vAh strain ML09-119, and relative expression of thirteen immune-related genes was evaluated from head kidney, spleen, and liver. Our results revealed that vAh was detected 2 hours post-infection (hpi) in the head kidney, liver, and spleen. The highest concentration of vAh was detected at 12 hpi, from which point concentrations decreased until clearance at 5 days post-infection (dpi). Gene expression analysis revealed upregulation of pro-inflammatory cytokines and innate immune response (TLR 4 and 5) in the first 24 hpi. Adaptive immune-related genes were upregulated at 7 dpi in the spleen and 14 dpi in the head kidney. Furthermore, immunoglobulin M showed significant upregulation at 14 dpi in the head kidney and 21 dpi in the spleen. In summary, vAh ML09-119 infection induced a strong inflammatory response involving multiple innate immunity genes, proinflammatory cytokines, and chemokines. Surviving catfish were able to clear the infection and produce antibodies and memory cells. Assessment of the immunological response to vAh infection is critical for understanding the pathogen's mechanisms of pathogenesis and developing means for MAS control, including vaccine development and improved treatments.

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.

Tad pili contribute to the virulence and biofilm formation of virulent Aeromonas hydrophila.

Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila, a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections.

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 invivo 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.

Biologic and genomic characterization of a novel virulent Aeromonas hydrophila phage phiA051, with high homology to prophages.

Aeromonas hydrophila is particularly harmful to freshwater aquaculture, and the search for phage is an effective biological control method, but reports of possible temperate phages and their mutants are rare in this field. In this study, a virulent phage highly homologous to prophage in the genomes of A. hydrophila was collected and preliminary biological characterization was carried out to understand its nature. Water samples taken from eel ponds in Fujian, China were combined with the strain. Spot test method and double-layer agar plate assay was used for confirmation and purification. Phage virions were observed using transmission electron microscope. A total of 68 strains of Aeromonas spp. were used to determine the host range. MOI groups of 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 were prepared to detect the optimal MOI. The conditions of thermal stability assay were set as 30, 40, 50, 60, 70 and 80°C for 1 h, respectively, and conditions of acid and alkali stability assay were set as 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 of pH. MOI of 0.01 and 0.1, respectively, are set to determine the inhibitory capacity of phage. A novel virulent A. hydrophila phage designated phiA051 has been isolated from aquaculture water. Electron microscopic observation showed that the phage phiA051 was composed of an icosahedral capsid. The phage phiA051 possesses an optimal multiplicity of infection (MOI) of 0.01, and its burst size was 108 PFU/cell. The phage maintained a high viability at temperatures of 30-50°C or pH 6.0-10.0 for 1 h. Phage phiA051 has certain potentials in rapidly inhibiting the spread of pathogen early in the outbreak, and it has a linear dsDNA with GC content of 60.55% and a total length of 32,212 bp, including 46 ORFs. The phage phiA051 behaved as a virulent phage. However, the BLASTN result showed that 23 of the top 25 hits were genomes of Aeromonas strains. It was suggested that phiA051 was probably derived from some prophage in the chromosome of Aeromonas. Further investigation of the mechanism how phage phiA051 transforms from a temperate phage to a virulent phage will provide a unique perspective and idea to explore the potential of prophages.

Unveiling a Virulence-Regulating Mechanism in Aeromonas hydrophila: a Quantitative Exoproteomic Analysis of an AraC-Like Protein.

Bacterial AraC is a transcription factor family that initiates transcription by recruiting RNA polymerase to the promoter and directly regulating various bacterial phenotypes. It also directly regulates various bacterial phenotypes. However, how this transcription factor regulates bacterial virulence and affects host immunity is still largely unknown. In this study, deleting the orf02889 (AraC-like transcription factor) gene in virulent Aeromonas hydrophila LP-2 affected several important phenotypes, such as increasing biofilm formation and siderophore production abilities. Moreover, Δorf02889 also significantly decreased the virulence of A. hydrophila and has promising attenuated vaccine potential. To better understand the effects of orf02889 on biological functions, a data independent acquisition (DIA)-based quantitative proteomics method was performed to compare the differentially expressed proteins between Δorf02889 and the wild-type strain in extracellular fractions. The following bioinformatics analysis suggested that ORF02889 may regulate various metabolic pathways, such as quorum sensing and ATP binding cassette (ABC) transporter metabolism. Moreover, 10 selected genes from the top 10 decreasing abundances in proteomics data were deleted, and their virulence to zebrafish was evaluated, respectively. The results showed that ΔcorC, Δorf00906, and Δorf04042 significantly reduced bacterial virulence. Finally, the following chromatin immunoprecipitation and polymerase chain reaction (ChIP-PCR) assay validated that the promoter of corC was directly regulated by ORF02889. Overall, these results provide insight into the biological function of ORF02889 and demonstrate its inherent regulatory mechanism for the virulence of A. hydrophila.

Development of Bioluminescent Virulent Aeromonas hydrophila for Understanding Pathogenicity.

Virulent Aeromonas hydrophila (vAh) strains that cause motile Aeromonas septicemia (MAS) in farmed channel catfish (Ictalurus punctatus) have been an important problem for more than a decade. However, the routes of infection of vAh in catfish are not well understood. Therefore, it is critical to study the pathogenicity of vAh in catfish. To this goal, a new bioluminescence expression plasmid (pAKgfplux3) with the chloramphenicol acetyltransferase (cat) gene was constructed and mobilized into vAh strain ML09-119, yielding bioluminescent vAh (BvAh). After determining optimal chloramphenicol concentration, plasmid stability, bacteria number-bioluminescence relationship, and growth kinetics, the catfish were challenged with BvAh, and bioluminescent imaging (BLI) was conducted. Results showed that 5 to 10 µg/mL chloramphenicol was suitable for stable bioluminescence expression in vAh, with some growth reduction. In the absence of chloramphenicol, vAh could not maintain pAKgfplux3 stably, with the half-life being 16 h. Intraperitoneal injection, immersion, and modified immersion (adipose fin clipping) challenges of catfish with BvAh and BLI showed that MAS progressed faster in the injection group, followed by the modified immersion and immersion groups. BvAh was detected around the anterior mouth, barbels, fin bases, fin epithelia, injured skin areas, and gills after experimental challenges. BLI revealed that skin breaks and gills are potential attachment and entry portals for vAh. Once vAh breaches the skin or epithelial surfaces, it can cause a systemic infection rapidly, spreading to all internal organs. To our best knowledge, this is the first study that reports the development of a bioluminescent vAh and provides visual evidence for catfish-vAh interactions. Findings are expected to provide a better understanding of vAh pathogenicity in 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.

Impacts of Fortifying Nile Tilapia (Oreochromis niloticus) Diet with Different Strains of Microalgae on Its Performance, Fillet Quality and Disease Resistance to Aeromonas hydrophila Considering the Interplay between Antioxidant and Inflammatory Response.

The oxidative stress facing fish during intensive production brings about diseases and mortalities that negatively influence their performance. Along with that, the increased awareness of omega-3 polyunsaturated fatty acids (omega-3-PUFAs) health benefits has been triggered the introduction of alternative additives in aqua feed that cause not only modulation in fish immune response but also fortification of their fillet. In this context, the role of microalgae mix (NSS) containing Nannochloropsis oculate and Schizochytrium and Spirulina species, which were enriched with bioactive molecules, especially EPA and DHA, was assessed on Nile tilapia's performance, fillet antioxidant stability, immune response, and disease resistance. Varying levels of NSS (0.75, 1.5, and 3%) were added to Nile tilapia's diet for 12 weeks and then a challenge of fish with virulent Aeromonas hydrophila (A. hydrophila) was carried out. Results showed that groups fed NSS, especially at higher levels, showed an improved WG and FCR, which corresponded with enhanced digestive enzymes' activities. Higher T-AOC was detected in muscle tissues of NSS3.0% fed fish with remarkable reduction in ROS, H2O2, and MDA contents, which came in parallel with upregulation of GSH-Px, CAT, and SOD genes. Notably, the contents of EPA and DHA in fillet were significantly increased with increasing the NSS levels. The mean log10 counts of pathogenic Vibrio and Staphylococcus species were reduced, and conversely, the populations of beneficial Lactobacillus and Bacillus species were increased more eminent after supplementation of NSS3.0% and NSS1.5%. Moreover, regulation of the immune response (lysozyme, IgM, ACH50, NO, and MPO), upregulation of IL-10, TGF-β, and IgM, and downregulation of IL-1β, TNF-α, HSP70,and COX-2 were observed following dietary higher NSS levels. After challenge, reduction in A. hydrophila counts was more prominent, especially in NSS3.0% supplemented group. Taken together, the current study encourages the incorporation of such microalgae mix in Nile tilapia's diet for targeting maximum performance, superior fillet quality, and protection against A. hydrophila.

Amphora coffeaeformis diatom involved in reducing the susceptibility of Nile tilapia ( Oreochromis niloticus ) fingerlings to Aeromonas hydrophila infection by boosting immune and antioxidant responses and improving growth performance indicators

In the present study, the Amphora coffeaeformis diatom was applied in different concentrations to the diet of Nile tilapia (Oreochromis niloticus) to investigate its potential effects in improving growth performance, as well as enhancing immune and antioxidant responses to control Aeromonas hydrophila infection. The tilapia fingerlings were divided into 4 groups, and for 4 weeks were fed A. coffeaeformis at various concentrations (0, 1, 2 and 3%). Growth-stimulating activity from dietary A. coffeaeformis was observed, particularly in the 2% and 3% treatment groups. Furthermore, serum immunoglobulin M (IgM), nitric oxide (NO), malondialdehyde (MDA), total-superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GPx) were evaluated at the 2nd and 4th weeks from the onset of feeding and showed significant improvement (p < 0.05) in the 2 and 3% groups. At the end of the feeding trial, the tilapia were challenged via an intraperitoneal (IP) injection of virulent Aeromonas hydrophila. Fish survival, serum IgM, nitric oxide, MDA, T-SOD, CAT, GPx, and liver, kidney, spleen, and gill histopathology were all evaluated post challenge. Significant improvements in both immunological and antioxidant parameters continued until the post-challenge measurements. Overall survival was significantly increased in the 2 and 3% groups, compared to the control and 1% groups. Histopathological examination revealed a decrease in the degenerative and destructive effects of A. hydrophila infection on the liver, kidneys, spleen, and gill tissues, indicating that dietary A. coffeaeformis administered at concentrations of 2 and 3% could be recommended as a feed additive for the control of A. hydrophila infection in Nile tilapia.

Identification and Characterization of Differentially Expressed IgM Transcripts of Channel Catfish Vaccinated with Antigens of Virulent Aeromonas hydrophila

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish.

Bactericidal efficacy of non-thermal plasma activation against Aeromonas hydrophila and immunological responses of koi (Cyprinus carpio haematopterus)

In the aquaculture industry, an efficient and safe water purification system is important to prevent mass mortality by virulent pathogens. As extensive use of traditional methods (e.g.: povidone-iodine, ozone, ultraviolet irradiation, formalin, and chlorine dioxide) have adverse effects on cultured fish, an appropriate and alternative water purification method is vital for the sustainability of the industry. Non-thermal plasma technology has been successfully used for various biomedical purposes (e.g: food sterilization, medical device disinfection, wound healing, cancer therapy, etc.) and has great potential to be used as a sterilizing system. However, few studies have been conducted on its usefulness in the aquaculture industry. In this study, we investigated the bactericidal efficacy of plasma-activated water induced by non-thermal plasma and its histopathological as well as immunological adverse effects on koi. A highly virulent Aeromonas hydrophila SNU HS7, which caused massive mortality of koi, was used for this study. Non-thermal plasma was applied for 10 min to the fish tanks with 1.2 × 109 CFU/mL SNU HS7 using PLMB-20 system to confirm the sterilization efficacy and to observe the survival and immunological reaction of koi for 14 days. As a result, gross pathological, histopathological, and immunological investigations did not reveal any significant adverse effects in fish as compared to the control groups. To the best of our knowledge, this is the first study showing that non-thermal plasma can be used for sterilization of rearing water without giving significant physiological damage to the fish, even under the assumption of extreme situations. As plasma can effectively sterilize not only bacteria but also other unknown pathogens, the results of this study are showing a promising future in purifying water in aquaculture practice.

Molecular detection and genetic diversity of virulent Aeromonas hydrophila (Chester, 1901) Stanier, 1943 isolates from raw and processed fish

Molecular detection and genetic diversity of virulent Aeromonas hydrophila (Chester, 1901) Stanier, 1943 isolates from raw and processed fish

Effect of dietary mannan oligosaccharide (MOS) on growth, digestive enzymes activity, innate immunity and disease resistance against virulent Aeromonas hydrophila in peninsular carp Barbodes carnaticus (Jerdon, 1849)

Effect of dietary mannan oligosaccharide (MOS) on growth, digestive enzymes activity, innate immunity and disease resistance against virulent Aeromonas hydrophila in peninsular carp Barbodes carnaticus (Jerdon, 1849)

Pathological findings of Chinese sucker (Myxocyprinus asiaticus) infected with virulent Aeromonas hydrophila

A highly virulent strain of Aeromonas hydrophila (vAh) has caused the motile Aeromonas septicemia (MAS) in Chinese sucker (Myxocyprinus asiaticus). The pathology of MAS in Chinese sucker has not been studies well yet. Thus, the aim of this study was to determine histopathological and ultrastructural changes in Chinese sucker following vAh challenge. Histopathological examination revealed hemorrhage, hemolysis, endothelial cell damage, necrosis, cell vacuolation, lymphocyte infiltration and MMCs response. The ultrastructure showed severe tissue destruction, such as the malformation of glomerulus filtration device in trunk kidney, macrophages with large phagocytic vacuoles in head kidney, reticular cells and endothelial cells necrosis in spleen, the disappearing of glycogen and the degeneration of the diss space in liver. In conclusion, these results indicated that vAh infection would lead to serious pathological changes in tissues, which maybe results in death by anoxia, energy shortage and metabolic waste accumulation. Moreover, we found that MMCs in tissues might play an important role in fighting vAh infection, which would provide a new perspective for studies on the pathomechanism and enrich the histopathological data on MAS.

Lactobacillus paraplantarum L34b-2 derived from fermented food improves the growth, disease resistance and innate immunity in Pangasius bocourti

Probiotics have increasingly gained interest as alternatives to antibiotics in controlling infectious diseases in aquaculture. This research aimed to isolate and evaluate lactic acid bacteria (LAB) for potential use as probiotics for Pangasius bocourti. A total of 656 LAB isolates were obtained from fish gut and fermented beef samples, of which 367 isolates displayed antagonistic activity against Aeromonas hydrophila FW52 and/or Streptococcus agalactiae F3S which were employed as indicator fish pathogens. Among these antagonistic LAB, only 18 isolates produced bacteriocin-like activity. These were further evaluated in vitro for other probiotic properties. After in vitro screening, the isolate L34b-2 obtained from Thai indigenous fermented beef was chosen as a suitable probiotic LAB for in vivo studies based on its remarkable probiotic characteristics. These included broad-spectrum bacteriocin-like activity, tolerance to simulated gastrointestinal tract conditions, mucin adhesion ability, co-aggregation ability with fish pathogens tested, non-blood hemolysis, antibiotic susceptibility and protease enzyme production. Identification of the isolate L34b-2 using conventional methods together with 16S rDNA analysis revealed that it belonged to Lactobacillus paraplantarum. Then, the effects of 60-day-dietary administration of L. paraplantarum L34b-2 on fish growth, innate immune responses and disease resistance were investigated in Pangasius bocourti. In feed-trial studies, after feeding for both 30 and 60 days it was found that the Pangasius fish fed with basal diet containing 107 CFU g−1 L. paraplantarum L34b-2 had significantly (p < 0.05) higher weight gain and specific growth rate, and the lowest feed conversion ratio, compared to the control fish that received only basal diet. For immune response studies, it was found that the fish fed with the probiotic strain L34b-2, when compared to the control fish, had significantly higher lysozyme, alternative complement and bactericidal activities but no significant differences (p > 0.05) in phagocytic and respiratory burst activities. After 60-day feeding, fish were subjected to challenge tests by intraperitoneal injection with 106 CFU of a virulent Aeromonas hydrophila FW52 and rearing was continued for two more weeks. As expected, fish that received L34b-2 had a significantly higher (p < 0.05) post challenge survival rate than the control group. These results clearly show that L. paraplantarum L34b-2 displayed beneficial effects on P. bocourti as a probiotic by which it not only promoted growth but also enhanced innate immunity and disease resistance against A. hydrophila.

Isolation and characterization of bacteriophages against virulent Aeromonas hydrophila

BackgroundAeromonas hydrophila is an important water-borne pathogen that leads to a great economic loss in aquaculture. Along with the abuse of antibiotics, drug-resistant strains rise rapidly. In addition, the biofilms formed by this bacterium limited the antibacterial effect of antibiotics. Bacteriophages have been attracting increasing attention as a potential alternative to antibiotics against bacterial infections.ResultsFive phages against pathogenic A. hydrophila, named N21, W3, G65, Y71 and Y81, were isolated. Morphological analysis by transmission electron microscopy revealed that phages N21, W3 and G65 belong to the family Myoviridae, while Y71 and Y81 belong to the Podoviridae. These phages were found to have broad host spectra, short latent periods and normal burst sizes. They were sensitive to high temperature but had a wide adaptability to the pH. In addition, the phages G65 and Y81 showed considerable bacterial killing effect and potential in preventing formation of A. hydrophila biofilm; and the phages G65, W3 and N21 were able to scavenge mature biofilm effectively. Phage treatments applied to the pathogenic A. hydrophila in mice model resulted in a significantly decreased bacterial loads in tissues.ConclusionsFive A. hydrophila phages were isolated with broad host ranges, low latent periods, and wide pH and thermal tolerance. And the phages exhibited varying abilities in controlling A. hydrophila infection. This work presents promising data supporting the future use of phage therapy.

The severity of motile Aeromonas septicemia caused by virulent Aeromonas hydrophila in channel catfish is influenced by nutrients and microbes in water

Motile Aeromonas septicemia (MAS), caused by virulent Aeromonas hydrophila (vAh), has severely impacted catfish farming in the southeastern United States since 2009. Conditions that trigger the onset of MAS outbreaks remain largely unknown. In this study, the effects of nutrients and select microbes in water on proliferation of vAh and severity of MAS in channel catfish were assessed. Results of the study demonstrated that both nutrient-rich tryptic soy broth powder (TSBp, the microbiological growth medium) and the commercial fish feed supported vigorous growth of vAh in water. By addition of 6 g TSBp or 6 g fish feed to 15 L water (0.04%, w/v), vAh multiplied from 2.5 × 105 CFU/mL to approximately 4.7 × 108 CFU/mL and 2.0 × 108 CFU/mL, respectively, at 24 h post inoculation (hpi). Challenge of fish in the vAh-propagated water at 24 hpi resulted in approximately 96% and 73% mortality, respectively. Co-inoculation of vAh with a vAh-antagonistic Pseudomonas mosselii or a non-antagonistic Aeromonas veronii in water had no significant effects on vAh growth and infection; nevertheless, P. mosselii protected fish against vAh infection (with approximately 79% of relative percentage of survival) when fish were briefly pre-immersed in P. mosselii-propagated water prior to challenge. Culture of vAh in situ described in this study was shown to be a useful method for mimicking vAh growth dynamics in response to nutrients and probiotics in relatively natural environment. Findings of this study would help understand the mechanism of MAS outbreaks and facilitate research on control and prevention of MAS.

Recombinant ATPase of Virulent Aeromonas hydrophila Protects Channel Catfish Against Motile Aeromonas Septicemia.

Channel catfish farming dominates the aquaculture industry in the United States. However, epidemic outbreaks of motile Aeromonas septicemia (MAS), caused by virulent Aeromonas hydrophila (vAh), have become a prominent problem in the catfish industry. Although vaccination is an effective preventive method, there is no vaccine available against MAS. Recombinant proteins could induce protective immunity. Thus, in this work, vAh ATPase protein was expressed, and its protective capability was evaluated in catfish. The purified recombinant ATPase protein was injected into catfish, followed by experimental infection with A. hydrophila strain ML09-119 after 21 days. Results showed catfish immunized with ATPase exhibited 89.16% relative percent survival after challenge with A. hydrophila strain ML09-119. Bacterial concentrations in liver, spleen, and anterior kidney were significantly lower in vaccinated fish compared with the non-vaccinated sham group at 48 h post-infection (p < 0.05). Catfish immunized with ATPase showed a significant (p < 0.05) higher antibody response compared to the non-vaccinated groups. Overall, ATPase recombinant protein has demonstrated potential to stimulate protective immunity in catfish against virulent A. hydrophila infection.

Direct Economic Impact of Fish Diseases on the East Mississippi Catfish Industry

AbstractThe objectives of this effort were to identify basic descriptors of the east Mississippi (MS) catfish industry, identify prevalent diseases affecting this region, and determine the direct economic impacts of these diseases representing the 2016 production season. Data from east MS catfish operations were collected over a 3‐month period during the late spring through early summer of 2017. A 95% confidence interval (CI) with finite correction was used to make inferences on the population. The number of operations (115) and number of hectares used for catfish production (4,900 ha) were identified by personal communications and confirmed by satellite imagery. Producers were surveyed a second time by e‐mail (29.6% response) to identify basic industry descriptors, the most predominant catfish diseases of east MS, and their respective direct economic impact. The top‐three diseases, as reported by producers, were virulent Aeromonas hydrophila, the “other” disease category, and Flavobacterium columnare. Producers were surveyed a third time by e‐mail (34% response) to identify the mean (±95% CI) of regional catfish production (8,070 ± 783 kg/ha). Foregone sales loss attributed to catfish disease was reported as the sum of fish loss ($10 million) and production loss ($4.3 million). Foregone income loss resulted from the increased expenditures associated with catfish disease and included medicated feed ($1.9 million), chemical applications ($0.6 million), and miscellaneous ($0.1 million). The total direct economic impact of catfish diseases for the 2016 east MS production season was reported as the sum of foregone sales and foregone income and totaled $16.9 million.