RESUMO
Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qß 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.
Assuntos
Biofilmes , Peixes-Gato , Edwardsiella ictaluri , Infecções por Enterobacteriaceae , Fator Proteico 1 do Hospedeiro , Edwardsiella ictaluri/genética , Edwardsiella ictaluri/patogenicidade , Animais , Fator Proteico 1 do Hospedeiro/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Biofilmes/crescimento & desenvolvimento , Infecções por Enterobacteriaceae/microbiologia , Peixes-Gato/microbiologia , Doenças dos Peixes/microbiologia , Virulência , Macrófagos/microbiologia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Células Epiteliais/microbiologia , Aderência Bacteriana/genéticaRESUMO
The interleukin-12 (IL-12) family is a class of heterodimeric cytokines that play crucial roles in pro-inflammatory and pro-stimulatory responses. Although some IL-12 and IL-23 paralogues have been found in fish, their functional activity in fish remains poorly understood. In this study, Pf_IL-12p35a/b, Pf_IL-23p19 and Pf_IL-12p40a/b/c genes were cloned from yellow catfish (Pelteobagrus fulvidraco), four α-helices were found in Pf_IL-12p35a/b and Pf_IL-23p19. The transcripts of these six genes were relatively high in mucus and immune tissues of healthy individuals, and in gill leukocytes. Following Edwardsiella ictaluri infection, Pf_IL-12p35a/b and Pf_IL-23p19 mRNAs were induced in brain and kidney (or head kidney), Pf_IL-12p40a mRNA was induced in gill, and Pf_IL-12p40b/c mRNAs were induced in brain and liver (or skin). The mRNA expression of these genes in PBLs was induced by phytohaemagglutinin (PHA) and polyinosinic-polycytidylic acid (poly I:C), while lipopolysaccharides (LPS) induced the mRNA expression of Pf_IL-12p35a and Pf_IL-12p40b/c in PBLs. After stimulation with recombinant (r) Pf_IL-12 and rPf_IL-23 subunit proteins, either alone or in combination, mRNA expression patterns of genes related to T helper cell development exhibited distinct differences. The results suggest that Pf_IL-12 and Pf_IL-23 subunits may play important roles in regulating immune responses to pathogens and T helper cell development.
Assuntos
Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Proteínas de Peixes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imunidade Inata , Subunidade p40 da Interleucina-12 , Animais , Peixes-Gato/genética , Peixes-Gato/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Perfilação da Expressão Gênica/veterinária , Imunidade Inata/genética , Edwardsiella ictaluri/fisiologia , Subunidade p35 da Interleucina-12/genética , Subunidade p35 da Interleucina-12/imunologia , Filogenia , Sequência de Aminoácidos , Alinhamento de Sequência/veterinária , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/imunologia , Poli I-C/farmacologiaRESUMO
To prevent catfish idiopathic anaemia, diets fortified with iron have been adopted as a regular practice on commercial catfish farms to promote erythropoiesis. However, the effects of prolonged exposure of excess dietary iron on production performance and disease resistance for hybrid catfish (Ictalurus punctatus × I. furcatus) remains unknown. Four experimental diets were supplemented with ferrous monosulphate to provide 0, 500, 1000, and 1500 mg of iron per kg of diet. Groups of 16 hybrid catfish juveniles (~22.4 g) were stocked in each of 20, 110-L aquaria (n = 5), and experimental diets were offered to the fish to apparent satiation for 12 weeks. At the end of the study, production performance, survival, condition indices, as well as protein and iron retention were unaffected by the dietary treatments. Blood haematocrit and the iron concentration in the whole-body presented a linear increase with the increasing the dietary iron. The remaining fish from the feeding trial was challenged with Edwardsiella ictaluri. Mortality was mainly observed for the dietary groups treated with iron supplemented diets. The results for this study suggest that iron supplementation beyond the required levels does affect the blood production, and it may increase their susceptibility to E. ictaluri infection.
Assuntos
Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Ictaluridae , Animais , Resistência à Doença , Edwardsiella ictaluri , Ferro/farmacologia , Ferro da Dieta , Hematócrito , Doenças dos Peixes/prevenção & controle , Dieta/veterinária , Suplementos Nutricionais , Infecções por Enterobacteriaceae/prevenção & controle , Infecções por Enterobacteriaceae/veterináriaRESUMO
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.
Assuntos
Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Microbioma Gastrointestinal , Ictaluridae , Probióticos , Tianfenicol/análogos & derivados , Animais , Edwardsiella ictaluri/fisiologia , Prebióticos , Disbiose , RNA Ribossômico 16S , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/prevenção & controle , Doenças dos Peixes/microbiologia , Antibacterianos/farmacologia , Suplementos Nutricionais , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/prevenção & controle , Infecções por Enterobacteriaceae/veterináriaRESUMO
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.
Assuntos
Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Ictaluridae , Sistemas de Secreção Tipo VI , Animais , Edwardsiella ictaluri/genética , Sistemas de Secreção Tipo VI/genética , Virulência , Doenças dos Peixes/prevenção & controleRESUMO
IMPORTANCE: This work has global significance in the catfish industry, which provides food for increasing global populations. E. ictaluri is a leading cause of disease loss, and EseN is an important player in E. ictaluri virulence. The E. ictaluri T3SS effector EseN plays an essential role in establishing infection, but the specific role EseN plays is not well characterized. EseN belongs to a family of phosphothreonine lyase effectors that specifically target host mitogen activated protein kinase (MAPK) pathways important in regulating host responses to infection. No phosphothreonine lyase equivalents are known in eukaryotes, making this family of effectors an attractive target for indirect narrow-spectrum antibiotics. Targeting of major vault protein and PDK1 kinase by EseN has not been reported in EseN homologs in other pathogens and may indicate unique functions of E. ictaluri EseN. EseN targeting of PDK1 is particularly interesting in that it is linked to an extraordinarily diverse group of cellular functions.
Assuntos
Infecções por Enterobacteriaceae , Doenças dos Peixes , Liases , Animais , Edwardsiella ictaluri/fisiologia , Fosfotreonina , Sistema de Sinalização das MAP Quinases , Macrófagos , Morte CelularRESUMO
Bacillary necrosis of pangasius (BNP) is a disease caused by Edwardsiella ictaluri bacteria in striped catfish Pangasianodon hypophthalmus that results in high mortality rates. To control this disease, bacteriophages have been considered as alternatives to antibiotics. In this study, we applied the lytic bacteriophage PVN06 in striped catfish fingerlings to prevent E. ictaluri infection. In an experimental trial, the phage was administered to fish by feeding phage-coated feed with doses of 7.17±0.09, 8.17±0.09 and 9.17±0.09 log PFU/g feed per day before bacterial infection. Fish were infected by bacteria once with concentrations ranging from 3.01 to 7.01 log CFU/ml tank water. A day after infection, phage treatment resumed at a rate of once per day until the end of the trial. The results of the trial show that bacterial infection caused typical symptoms of BNP in fish with the cumulative fish death rate of 36.7±2.9 to 75.0±5.0%, depending on the bacterial concentration used for infection. Phage treatment with 9.17±0.09 log PFU/g significantly reduced the mortality rate, while treatments with 8.17±0.09 and 7.17±0.09 log PFU/g did not. This phage dose resulted in a 61.7-fold reduction in the toxicity of the bacterial pathogen and the survival rate of 15-23.3% in fish. Our study has demonstrated that the bacteriophage PVN06 protected striped catfish from BNP.
Assuntos
Bacteriófagos , Peixes-Gato , Infecções por Enterobacteriaceae , Animais , Infecções por Enterobacteriaceae/prevenção & controle , Edwardsiella ictaluriRESUMO
Widespread distribution of a highly pathogenic Edwardsiella ictaluri strain in farmed tilapia in northern Vietnam has recently been reported. The subsequent investigation noticed a disease outbreak occurred at five nearby tilapia farms with floating cages, in which the clinical signs of both edwardsiellosis and columnaris diseases were observed on the same infected fish and caused 65% to 85% fish mortality. Naturally diseased fish (n = 109) were sampled from the five infected farms for bacterial identification and conducting challenge tests. The two bacteria Edwardsiella ictaluri and Flavobacterium oreochromis were identified by a combination of biochemical tests, PCR and 16SrRNA sequencing methods. Experimental challenge tests on Nile tilapia resulted in the median lethal dose (LD50 ) of E. ictaluri and F. oreochromis at 70 CFU/fish by intraperitoneal (i.p.) injection and 3.6 × 106 CFU/mL by immersion, respectively. The experimentally co-infected challenged fish exposed to LD50 doses resulted in 83% ± 6% mortality, with the infected fish exhibiting clinical signs of both edwardsiellosis and columnaris diseases, mimicking the naturally diseased fish. This finding suggests that the co-infection of E. ictaluri and F. oreochromis may interact in a synergistic manner, to enhance the overall severity of the infection and elevates the need for efficient methods to control both pathogens.
Assuntos
Ciclídeos , Infecções por Enterobacteriaceae , Doenças dos Peixes , Tilápia , Animais , Edwardsiella ictaluri/genética , Flavobacterium , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/veterinária , Infecções por Enterobacteriaceae/microbiologia , Doenças dos Peixes/microbiologiaRESUMO
The yellow catfish (Pelteobagrus fulvidraco) is an economic fish with a large breeding scale, and diseases have led to huge economic losses. Tumor necrosis factor receptor-associated factors (TRAFs) are a class of intracellular signal transduction proteins that play an important role in innate and adaptive immune responses by mediating NF-κB, JNK and MAPK signaling pathways. However, there are few studies on the TRAF gene family in yellow catfish. In this study, the open reading frame (ORF) sequences of TRAF1, TRAF2a, TRAF2b, TRAF3, TRAF4a, TRAF4b, TRAF5, TRAF6 and TRAF7 genes were cloned and identified in yellow catfish. The ORF sequences of the nine TRAF genes of yellow catfish (Pf_TRAF1-7) were 1413-2025 bp in length and encoded 470-674 amino acids. The predicted protein structures of Pf_TRAFs have typically conserved domains compared to mammals. The phylogenetic relationships showed that TRAF genes are conserved during evolution. Gene structure, motifs and syntenic analyses of TRAF genes showed that the exon-intron structure and conserved motifs of TRAF genes are diverse among seven vertebrate species, and the TRAF gene family is relatively conserved evolutionarily. Among them, TRAF1 is more closely related to TRAF2a and TRAF2b, and they may have evolved from a common ancestor. TRAF7 is quite different and distantly related to other TRAFs. Real-time quantitative PCR (qRT-PCR) results showed that all nine Pf_TRAF genes were constitutively expressed in 12 tissues of healthy yellow catfish, with higher mRNA expression levels in the gonad, spleen, brain and gill. After infection with Edwardsiella ictaluri, the expression levels of nine Pf_TRAF mRNAs were significantly changed in the head kidney, spleen, gill and brain tissues of yellow catfish, of which four genes were down-regulated and one gene was up-regulated in the head kidney; four genes were up-regulated and four genes were down-regulated in the spleen; two genes were down-regulated, one gene was up-regulated, and one gene was up-regulated and then down-regulated in the gill; one gene was up-regulated, one gene was down-regulated, and four genes were down-regulated and then up-regulated in the brain. These results indicate that Pf_TRAF genes might be involved in the immune response against bacterial infection. Subcellular localization results showed that all nine Pf_TRAFs were found localized in the cytoplasm, and Pf_TRAF2a, Pf_TRAF3 and Pf_TRAF4a could also be localized in the nucleus, uncovering that the subcellular localization of TRAF protein may be closely related to its structure and function in cellular mechanism. The results of this study suggest that the Pf_TRAF gene family plays important roles in the immune response against pathogen invasion and will provide basic information to further understand the roles of TRAF gene against bacterial infection in yellow catfish.
Assuntos
Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Animais , Edwardsiella ictaluri/metabolismo , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/veterinária , Peixes-Gato/genética , Fator 1 Associado a Receptor de TNF/genética , Fator 1 Associado a Receptor de TNF/metabolismo , Filogenia , Fator 3 Associado a Receptor de TNF/genética , Proteínas de Peixes/metabolismo , Mamíferos/metabolismoRESUMO
OBJECTIVES: Edwardsiella ictaluri is an important pathogen in farmed raised catfish. Recently, we showed that resistance to tetracycline and florfenicol in the E. ictaluri MS-17-156 strain isolated from channel catfish was facilitated by acquisition of a 135 kb plasmid (named pEIMS-171561). METHODS: We described the genetic structure of pEIMS-171561. Plasmid copy number and stability within E. ictaluri strain MS-17-156 was determined. We also investigated the in vitro and in vivo transferability of pEIMS-171561 using catfish as a model for in vivo transfer. RESULTS: pEIMS-171561 belonged to the IncA/C group and contained florfenicol efflux major facilitator superfamily (MFS) (floR), sulfonamides (sul2), and tetracycline efflux MFS (tetD) genes. The plasmid contained two conjugative transfer-associated regions and encoded six transposases and insertion sequences. In vitro conjugation experiments demonstrated that the IncA/C plasmid can transfer from E. ictaluri to Escherichia coli. The plasmid was stable in E. ictaluri without selection pressure for 33 days. We showed that pEIMS-171561 did not transfer from E. ictaluri MS-17-156 to endogenous microbiota in catfish. Moreover, we could not detect in vivo conjugal transfer of pEIMS-171561 from E. ictaluri to E. coli. Results from real-time PCR revealed upregulation of the floR gene in the intestines of catfish receiving florfenicol-medicated feed, compared with that in catfish receiving unmedicated feed. CONCLUSION: This study demonstrated that pEIMS-171561 did not disseminate from E. ictaluri to gut microbiota under selective pressure. This result suggests a limited role of the fish microbiota as a reservoir for this plasmid and for the spread of resistance.
Assuntos
Peixes-Gato , Infecções por Enterobacteriaceae , Animais , Edwardsiella ictaluri/genética , Escherichia coli/genética , Infecções por Enterobacteriaceae/veterinária , Infecções por Enterobacteriaceae/tratamento farmacológico , Plasmídeos/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Tetraciclina/uso terapêutico , Peixes-Gato/genética , Resistência Microbiana a MedicamentosRESUMO
The rifampicin-resistant strain E9-302 of Edwardsiella ictaluri strain 669 (WT) was generated by continuous passage on BHI agar plates containing increasing concentrations of rifampicin. E9-302 was attenuated significantly by 119 times to zebrafish Danio rerio compared to WT in terms of the 50% lethal dose (LD50). Zebrafish vaccinated with E9-302 via intraperitoneal (IP) injection at a dose of 1 × 103 CFU/fish had relative percentage survival (RPS) rates of 85.7% when challenged with wild-type E. ictaluri via IP 14 days post-vaccination (dpv). After 14 days of primary vaccination with E9-302 via immersion (IM) at a dose of 4 × 107 CFU/ml, a booster IM vaccination with E9-302 at a dose of 2 × 107 CFU/ml exhibited 65.2% RPS against challenge with wild-type E. ictaluri via IP 7 days later. These results indicated that the rifampicin-resistant attenuated strain E9-302 had potential as a live vaccine against E. ictaluri infection. A previously unreported amino acid site change at position 142 of the RNA polymerase (RNAP) ß subunit encoded by the gene rpoB associated with rifampicin resistance was identified. Analysis of the whole-genome sequencing results revealed multiple missense mutations in the virulence-related genes esrB and sspH2 in E9-302 compared with WT, and a 189 bp mismatch in one gene, whose coding product was highly homologous to glycosyltransferase family 39 protein. This study preliminarily explored the molecular mechanism underlying the virulence attenuation of rifampicin-resistant strain E9-302 and provided a new target for the subsequent study of the pathogenic mechanism of E. ictaluri.
Assuntos
Infecções por Enterobacteriaceae , Doenças dos Peixes , Animais , Infecções por Enterobacteriaceae/prevenção & controle , Edwardsiella ictaluri/genética , Peixe-Zebra , Rifampina , Virulência , Vacinas Atenuadas , Doenças dos Peixes/prevenção & controle , Vacinas BacterianasRESUMO
Catfish farming is the largest aquaculture industry in the United States and an important economic driver in several southeastern states. Edwardsiella piscicida is a Gram-negative pathogen associated with significant losses in catfish aquaculture. Several Gram-negative bacteria use the BasS/BasR two-component system (TCS) to adapt to environmental changes and the host immune system. Currently, the role of BasS/BasR system in E. piscicida virulence has not been characterized. In the present study, two mutants were constructed by deleting the basS and basR genes in E. piscicida strain C07-087. Both mutant strains were characterized for virulence and immune protection in catfish hosts. The EpΔbasS and EpΔbasR mutants were more sensitive to acidic environments and produced significantly less biofilm than the wild-type. In vivo studies in channel catfish (Ictalurus punctatus) revealed that both EpΔbasS and EpΔbasR were significantly attenuated compared with the parental wild-type (3.57% and 4.17% vs. 49.16% mortalities). Moreover, there was significant protection, 95.2% and 92.3% relative percent survival (RPS), in channel catfish vaccinated with EpΔbasS and EpΔbasR against E. piscicida infection. Protection in channel catfish was associated with a significantly higher level of antibodies and upregulation of immune-related genes (IgM, IL-8 and CD8-α) in channel catfish vaccinated with EpΔbasS and EpΔbasR strains compared with non-vaccinated fish. Hybrid catfish (channel catfish â × blue catfish â) challenges demonstrated long-term protection against subsequent challenges with E. piscicida and E. ictaluri. Our findings demonstrate BasS and BasR contribute to acid tolerance and biofilm formation, which may facilitate E. piscicida survival in harsh environments. Further, our results show that EpΔbasS and EpΔbasR mutants were safe and protective in channel catfish fingerlings, although their virulence and efficacy in hybrid catfish warrant further investigation. These data provide information regarding an important mechanism of E. piscicida virulence, and it suggests EpΔbasS and EpΔbasR strains have potential as vaccines against this emergent catfish pathogen.
Assuntos
Bass , Peixes-Gato , Edwardsiella , Infecções por Enterobacteriaceae , Doenças dos Peixes , Ictaluridae , Animais , Vacinas Bacterianas , Infecções por Enterobacteriaceae/prevenção & controle , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/microbiologia , Edwardsiella ictaluri/genéticaRESUMO
In the mid-2010s, Edwardsiella tarda was reaffiliated into three discrete taxa (E. anguillarum, E. piscicida, and E. tarda), obscuring previous descriptions of E. tarda-induced pathology in fish. To clarify ambiguity regarding the pathology of E. tarda, E. piscicida, and E. anguillarum infections in US farm-raised catfish, channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and channel × blue catfish hybrids were challenged with comparable doses of each bacterium. The most severe pathology and mortality occurred in fish challenged with E. piscicida, supporting previous reports of increased pathogenicity in commercially important ictalurids, while E. anguillarum and E. tarda warrant only minimal concern. Acute pathologic lesions among bacterial species were predominantly necrotizing and characteristic of gram-negative sepsis but became progressively granulomatous over time. After 100 days, survivors were exposed to the approximate median lethal doses of E. piscicida and E. ictaluri, revealing some cross-protective effects among E. piscicida, E. anguillarum, and E. ictaluri. In contrast, no fish that survived E. tarda challenge demonstrated any protection against E. piscicida or E. ictaluri. This work supports reports of increased susceptibility of channel, blue, and hybrid catfish to E. piscicida, while highlighting potential cross-protective affects among fish associated Edwardsiella spp.
Assuntos
Peixes-Gato , Edwardsiella , Infecções por Enterobacteriaceae , Doenças dos Peixes , Ictaluridae , Animais , Edwardsiella ictaluri , Edwardsiella tarda , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/microbiologia , VirulênciaRESUMO
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.
Assuntos
Peixes-Gato , Edwardsiella ictaluri , Infecções por Enterobacteriaceae , Doenças dos Peixes , Animais , Peixes-Gato/microbiologia , Edwardsiella ictaluri/patogenicidade , Edwardsiella ictaluri/ultraestrutura , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/microbiologia , HeminaRESUMO
Constructs bearing the cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (ß-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F3 channel catï¬sh family transgenic for cecropin transgene driven by the CMV promoter, and one F1 channel catï¬sh family transgenic for cecropin transgene driven by the common carp ß-actin promoter were produced. F3 and F1 individuals exhibited enhanced disease resistance when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). Inheritance of the transgene by the F1 and F3 generation was 15% and 60%, respectively. Growth rates of the cecropin transgenic and non-transgenic full siblings (controls) channel catfish were not different (P > 0.05). All transgenic fish showed significant resistance to infection by ESC at day 3 and day 4 post exposure (P = 0.005). No correlation was detected between body weight and time to death for all genetic groups (P = 0.34). Results of our study confirmed that genetic enhancement of E. ictaluri resistance can be achieved by cecropin transgenesis in channel catfish. In addition to survival rate, improving survival time is essential because the extension of survival time gives a better chance to apply treatments to stop the bacterial infection.
Assuntos
Peixes-Gato , Cecropinas , Infecções por Citomegalovirus , Infecções por Enterobacteriaceae , Doenças dos Peixes , Ictaluridae , Actinas/genética , Animais , Peixes-Gato/genética , Edwardsiella ictaluri/fisiologia , Infecções por Enterobacteriaceae/microbiologia , Doenças dos Peixes/microbiologia , Técnicas de Transferência de Genes , Ictaluridae/genética , Ictaluridae/microbiologiaRESUMO
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 (â¼20 × 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.
Assuntos
Vacinas Bacterianas , Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Animais , Aderência Bacteriana , Peixes-Gato/microbiologia , Edwardsiella ictaluri , Infecções por Enterobacteriaceae/prevenção & controle , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Imersão , Vacinas AtenuadasRESUMO
Edwardsiella piscicida is a growing problem for catfish aquaculture in the southeastern United States, particularly in channel (Ictalurus punctatus) x blue (I. furcatus) catfish hybrids. Research has shown E. piscicida isolates recovered from farmed catfish in Mississippi form at least five discrete phyletic groups, with no apparent differences in virulence in channel and hybrid catfish. Laboratory trials have shown a live-attenuated E. ictaluri vaccine (340X2) cross-protects against at least one E. piscicida isolate (S11-285) in channel and hybrid catfish, although it is unknown if this protection exists for other E. piscicida variants. To this end, channel and hybrid catfish were immunized by immersion with E. ictaluri 340X2. Thirty days later, fish were challenged by intracoelomic injection with representative E. piscicida variants from each phyletic group. Relative percent survival (RPS) for hybrids ranged from 54.7% to 77.8%, while RPS in channels ranged from 80.5% to 100%. A second study investigated whether channel and hybrid catfish exposed to heterologous E. piscicida isolates were similarly protected against wild-type E. ictaluri. Fish were exposed by bath immersion to representative E. piscicida isolates from each phyletic group. Thirty days post-immunization, fish were challenged by immersion with wild-type E. ictaluri isolate S97-773. Regardless of variant, previous exposure to heterologous E. piscicida isolates significantly improved survival following E. ictaluri challenge. These findings suggest the presence of shared and conserved antigens among E. piscicida and E. ictaluri that could be exploited by application of polyvalent or cross-protective vaccines.
Assuntos
Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Ictaluridae , Animais , Edwardsiella , Edwardsiella ictaluri , Infecções por Enterobacteriaceae/prevenção & controle , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/prevenção & controle , Vacinas AtenuadasRESUMO
Edwardsiella ictaluri is an emerging bacterial pathogen that affects farmed tilapia (Oreochromis spp.). This study reports the widespread presence of E. ictaluri in farmed tilapia in Vietnam. Among 26 disease outbreaks from nine provinces in Northern Vietnam during 2019-2021, 19 outbreaks originated from imported seeds, while outbreaks in seven farms were from domestic sources. Clinically sick fish showed the appearance of numerous white spots in visceral organs, and accumulative mortality reached 30%-65%. A total of 26 representative bacterial isolates recovered from 26 disease outbreaks were identified as E. ictaluri based on a combination of phenotypic tests, genus- and species-specific polymerase chain reaction assays, 16S rRNA and gyrB sequencing, and phylogenetic analysis. All isolates harboured the same virulence gene profiles esrC+ , evpC+ , ureA-C+ , eseI- , escD- and virD4- . Antimicrobial susceptibility tests revealed that 80.8%-100% of isolates were multidrug resistant, with resistance to 4-8 antimicrobials in the groups of penicillin, macrolides, sulfonamides, amphenicols and glycopeptides. The experimental challenge successfully induced disease that mimicked natural infection. The median lethal doses (LD50 ) of the tested isolates (n = 4) were 42-61 colony forming units/fish, indicating their extremely high virulence. This emerging pathogen is established and has spread to various geographical locations, causing serious impacts on farmed tilapia in northern Vietnam. It is likely that this pathogen will continue to spread through contaminated stocks (both imported and domestic sources) and persist. Thus, increased awareness, combined with biosecurity measures and emergent vaccination programs is essential to mitigate the negative impact of this emerging disease on the tilapia farming industry.
Assuntos
Ciclídeos , Infecções por Enterobacteriaceae , Doenças dos Peixes , Tilápia , Animais , Antibacterianos/farmacologia , Cloranfenicol , Ciclídeos/genética , Edwardsiella ictaluri/genética , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/microbiologia , Glicopeptídeos/genética , Macrolídeos , Penicilinas , Filogenia , RNA Ribossômico 16S/genética , Sulfonamidas , UreiaRESUMO
Identification of genetic markers associated with resistance against enteric septicemia of catfish (ESC) is of great interest for genetic enhancement programs of catfish. In the present study, bulk segregant RNA-Seq analysis was applied to determine differentially expressed genes and alleles after ESC infection. Here we report three genomic regions on LG1, LG12, and LG26, containing significant single-nucleotide polymorphisms (SNPs). These genomic regions aligned well with quantitative trait loci (QTL) previously identified. Within the QTL regions, eleven genes were found to be differentially regulated between phenotypic bulks. Importantly, the QTL on linkage group 1 (LG1) were found to be expressed in the liver, whereas the QTL on LG12 and LG26 were expressed in the intestine, suggesting multiple mechanisms of ESC resistance. It is apparent that apolipoproteins may be important for ESC resistance as the QTL on LG1 included the 14-kDa apolipoprotein genes that are both allelically expressed and differentially expressed between the resistant and susceptible bulks. Traf2 and NCK-interacting protein kinase (TNIK) were found in the QTL on LG12, and it was downregulated in resistant fish, suggesting the importance of NCK downregulation in ESC resistance, as previously reported. In addition, we observed divergent gene expression patterns between the liver and intestine after infection. Immune/inflammatory-related processes were overrepresented from liver DEGs, while those DEGs identified from intestine were enriched for proteolysis and wounding processes. Taken together, the BSR-Seq analysis presented here advanced the knowledge of ESC resistance, providing information of not only positions of QTL but also genes and their differential expression between resistant and susceptible fish, making it one step closer to the identification of the causal genes for ESC resistance.
Assuntos
Infecções por Enterobacteriaceae , Doenças dos Peixes , Ictaluridae , Animais , Edwardsiella ictaluri , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/genética , Ictaluridae/genética , RNA-SeqRESUMO
Assessments of genomic prediction accuracies using artificial intelligent (AI) algorithms (i.e., machine and deep learning methods) are currently not available or very limited in aquaculture species. The principal aim of this study was to examine the predictive performance of these new methods for disease resistance to Edwardsiella ictaluri in a population of striped catfish Pangasianodon hypophthalmus and to make comparisons with four common methods, i.e., pedigree-based best linear unbiased prediction (PBLUP), genomic-based best linear unbiased prediction (GBLUP), single-step GBLUP (ssGBLUP) and a nonlinear Bayesian approach (notably BayesR). Our analyses using machine learning (i.e., ML-KAML) and deep learning (i.e., DL-MLP and DL-CNN) together with the four common methods (PBLUP, GBLUP, ssGBLUP, and BayesR) were conducted for two main disease resistance traits (i.e., survival status coded as 0 and 1 and survival time, i.e., days that the animals were still alive after the challenge test) in a pedigree consisting of 560 individual animals (490 offspring and 70 parents) genotyped for 14,154 single nucleotide polymorphism (SNPs). The results using 6,470 SNPs after quality control showed that machine learning methods outperformed PBLUP, GBLUP, and ssGBLUP, with the increases in the prediction accuracies for both traits by 9.1-15.4%. However, the prediction accuracies obtained from machine learning methods were comparable to those estimated using BayesR. Imputation of missing genotypes using AlphaFamImpute increased the prediction accuracies by 5.3-19.2% in all the methods and data used. On the other hand, there were insignificant decreases (0.3-5.6%) in the prediction accuracies for both survival status and survival time when multivariate models were used in comparison to univariate analyses. Interestingly, the genomic prediction accuracies based on only highly significant SNPs (P < 0.00001, 318-400 SNPs for survival status and 1,362-1,589 SNPs for survival time) were somewhat lower (0.3-15.6%) than those obtained from the whole set of 6,470 SNPs. In most of our analyses, the accuracies of genomic prediction were somewhat higher for survival time than survival status (0/1 data). It is concluded that although there are prospects for the application of genomic selection to increase disease resistance to E. ictaluri in striped catfish breeding programs, further evaluation of these methods should be made in independent families/populations when more data are accumulated in future generations to avoid possible biases in the genetic parameters estimates and prediction accuracies for the disease-resistant traits studied in this population of striped catfish P. hypophthalmus.