Evaluation of Edwardsiella piscicida basS and basR mutants as vaccine candidates in catfish against edwardsiellosis.

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 ♀ × blue catfish ♂) 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.

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.

Edwardsiella ictaluri evpP is required for colonisation of channel catfish ovary cells and necrosis in anterior kidney macrophages.

Edwardsiella ictaluri is a Gram-negative facultative anaerobe that can survive inside channel catfish phagocytes. E. ictaluri can orchestrate Type VI Secretion System (T6SS) for survival in catfish macrophages. evpP encodes one of the T6SS translocated effector proteins. However, the role of evpP in E. ictaluri is still unexplored. In this work, we constructed an E. ictaluri evpP mutant (EiΔevpP) and assessed its survival under complement and oxidative stress. Persistence of EiΔevpP in catfish as well as attachment and invasion in catfish macrophage and ovary cells were determined. Further, virulence of EiΔevpP in catfish and apoptosis it caused in macrophages were explored. EiΔevpP behaved same as wild type (EiWT) under complement and oxidative stress in complex media, whereas oxidative stress affected mutant's survival significantly in minimal media (p < .05). Persistence of EiΔevpP in live catfish and uptake and survival inside peritoneal macrophages were similar. The attachment and invasion capabilities of EiΔevpP in catfish ovary cells were significantly less than that of EiWT (p < .05). Although EiΔevpP showed reduced attenuation in catfish, causing decreased catfish mortality compared with EiWT (44.73% vs. 67.53%), this difference was not significant. The apoptosis assay using anterior kidney macrophages indicated that the number of live macrophages exposed to EiΔevpP was significantly higher compared with EiWT exposed macrophages at 24-hr post-treatment (p < .05). However, there were no significant differences in the early and late apoptosis. Remarkably, necrosis in EiΔevpP exposed macrophages was significantly less than that of EiWT exposed macrophages at 24 hr (p < .05). Our results demonstrated that evpP is required for colonisation of catfish ovary cells and increased apoptosis and necrosis in anterior kidney macrophages.

Virulence and live vaccine potential of Edwardsiella piscicida phoP and phoQ mutants in catfish against edwardsiellosis.

Edwardsiella piscicida is a Gram-negative facultative intracellular bacterium causing edwardsiellosis in catfish, the largest aquaculture industry in the United States. A safe and effective vaccine is an urgent need to avoid economic losses associated with E. piscicida outbreaks. PhoP/PhoQ is a two-component signal transduction system (TCS) that plays an important role in bacterial pathogenesis through sense and response to environmental and host stress signals. This study aimed to explore the contribution of PhoQ/PhoP in E. piscicida virulence and develop live attenuated vaccines against E. piscicida infection in channel catfish (Ictalurus punctatus) and hybrid catfish (channel catfish ♀ × blue catfish (I. furcatus) ♂). In the current study, two in-frame deletion mutants were constructed by deleting phoP (ETAC_09785) and phoQ (ETAC_09790) genes in E. piscicida strain C07-087, and the virulence and protection efficacy of the constructed strains were evaluated in catfish following intraperitoneal injection. Both EpΔphoP and EpΔphoQ strains had a delayed adaptation to oxidative stress (0.2% H2 O2 ) compared to E. piscicida wild type. The EpΔphoP and EpΔphoQ mutants produced significantly less biofilm compared to wild-type E. piscicida. Notably, EpΔphoP and EpΔphoQ mutants were significantly attenuated in channel catfish compared with wild-type E. piscicida (6.63% and 4.17% versus 49.16% mortalities), and channel catfish vaccinated with EpΔphoP and EpΔphoQ were significantly protected (95.65% and 97.92% survival) against E. piscicida infection at 21 days post-vaccination. In hybrid catfish, EpΔphoP was significantly more attenuated than EpΔphoQ, but EpΔphoQ provided significantly better protection than EpΔphoP. EpΔphoP and EpΔphoQ strains both induced specific antibodies in channel catfish against E. piscicida at 14 and 21 days post-vaccination. This result indicated that EpΔphoP and EpΔphoQ mutants were safe and protective in channel catfish fingerlings, while EpΔphoP was safe in hybrid catfish. Our findings show that PhoP and PhoQ are required for adaptation to oxidative stress and biofilm formation and may help E. piscicida face tough environmental challenges; thus, functional PhoP and PhoQ are critical for a successful infection.

Contributions of a LysR Transcriptional Regulator to Listeria monocytogenes Virulence and Identification of Its Regulons.

The capacity of Listeria monocytogenes to adapt to environmental changes is facilitated by a large number of regulatory proteins encoded by its genome. Among these proteins are the uncharacterized LysR-type transcriptional regulators (LTTRs). LTTRs can work as positive and/or negative transcription regulators at both local and global genetic levels. Previously, our group determined by comparative genome analysis that one member of the LTTRs (NCBI accession no. WP_003734782) was present in pathogenic strains but absent from nonpathogenic strains. The goal of the present study was to assess the importance of this transcription factor in the virulence of L. monocytogenes strain F2365 and to identify its regulons. An L. monocytogenes strain lacking lysR (the F2365ΔlysR strain) displayed significant reductions in cell invasion of and adhesion to Caco-2 cells. In plaque assays, the deletion of lysR resulted in a 42.86% decrease in plaque number and a 13.48% decrease in average plaque size. Furthermore, the deletion of lysR also attenuated the virulence of L. monocytogenes in mice following oral and intraperitoneal inoculation. The analysis of transcriptomics revealed that the transcript levels of 139 genes were upregulated, while 113 genes were downregulated in the F2365ΔlysR strain compared to levels in the wild-type bacteria. lysR-repressed genes included ABC transporters, important for starch and sucrose metabolism as well as glycerolipid metabolism, flagellar assembly, quorum sensing, and glycolysis/gluconeogenesis. Conversely, lysR activated the expression of genes related to fructose and mannose metabolism, cationic antimicrobial peptide (CAMP) resistance, and beta-lactam resistance. These data suggested that lysR contributed to L. monocytogenes virulence by broad impact on multiple pathways of gene expression.IMPORTANCEListeria monocytogenes is the causative agent of listeriosis, an infectious and fatal disease of animals and humans. In this study, we have shown that lysR contributes to Listeria pathogenesis and replication in cell lines. We also highlight the importance of lysR in regulating the transcription of genes involved in different pathways that might be essential for the growth and persistence of L. monocytogenes in the host or under nutrient limitation. Better understanding L. monocytogenes pathogenesis and the role of various virulence factors is necessary for further development of prevention and control strategies.

Genomic diversity in flavobacterial pathogens of aquatic origin.

Flavobacterium species are considered important fish pathogens in wild and cultured fish throughout the world. They can cause acute, subacute, and chronic infections, which are mainly characterized by gill damage, skin lesions, and deep necrotic ulcerations. Primarily, three Flavobacterium species, F. branchiophilum, F. columnare, and F. psychrophilum, have been reported to cause substantial losses to freshwater fish. In this study, we evaluated genomes of 86 Flavobacterium species isolated from aquatic hosts (mainly fish) to identify their unique and shared genome features. Our results showed that F. columnare genomes cluster into four different genetic groups. In silico secretion system analysis identified that all genomes carry type I (T1SS) and type IX (T9SS) secretion systems, but the number of type I secretion system genes shows diversity between species. F. branchiophilum, F. araucananum, F. chilense, F. spartansii, and F. tructae genomes have full type VI secretion system (T6SS). F. columnare, F. hydatis, and F. plurextorum carry partial T6SS with some of the T6SS genes missing. F. columnare, F. araucananum, F. chilense, F. spartansii, F. araucananum, F. tructae, Flavobacterium sp., F. crassostreae, F. succinicans, F. hydatis, and F. plurextorum carry most of the type IV secretion system genes (T4SS). F. columnare genetic groups 1 and 2, Flavobacterium sp., and F. crassostreae encode the least number of antibiotic resistance elements. F. hydatis, F. chilense, and F. plurextorum encode the greatest number of antibiotic resistance genes. Additionally, F. spartansii, F. araucananum, and chilense encode the greatest number of virulence genes while Flavobacterium sp. and F. crassostreae encode the least number of virulence genes. In conclusion, comparative genomics of Flavobacterium species of aquatic origin will help our understanding of Flavobacterium pathogenesis.

An Edwardsiella piscicida esaS mutant reveals contribution to virulence and vaccine potential.

Edwardsiella piscicida is a Gram-negative pathogen that causes disease in diverse aquatic organisms. The disease leads to extensive losses in commercial aquaculture species, including farmed U.S. catfish. The type III secretion system (T3SS) often contributes to virulence of Gram-negative bacteria. The E. piscicida esaS gene encodes a predicted T3SS export apparatus protein. In the current study, an E. piscicida esaS mutant was constructed and characterized to increase our understanding of the role of T3SS in E. piscicida virulence. Deletion of esaS did not significantly affect biofilm formation and hemolytic activity of E. piscicida, but it had significant effects on expression of hemolysis and T3SS effector genes during biofilm growth. EpΔesaS showed significantly (P < 0.05) reduced virulence in catfish compared to the parent strain. No mortalities occurred in fish infected with EpΔesaS at 6.3 × 105 and 1.26 × 106 CFU/fish compared to 26% mortality in fish infected with wild-type E. piscicida at 7.5 × 105 CFU/fish. Bioluminescence imaging indicated that EpΔesaS invades catfish and colonizes for a short period in the organs. Furthermore, catfish immunized with EpΔesaS at 6.3 × 105 and 1.26 × 106 CFU provided 47% and 87% relative percent survival, respectively. These findings demonstrated that esaS plays a role in E. piscicida virulence, and the deletion mutant has vaccine potential for protection against wild-type E. piscicida infection.

Transposon mutagenesis and identification of mutated genes in growth-delayed Edwardsiella ictaluri.

BACKGROUND: Edwardsiella ictaluri is a Gram-negative facultative intracellular anaerobe and the etiologic agent of enteric septicemia of channel catfish (ESC). To the catfish industry, ESC is a devastating disease due to production losses and treatment costs. Identification of virulence mechanisms of E. ictaluri is critical to developing novel therapeutic approaches for the disease. Here, we report construction of a transposon insertion library and identification of mutated genes in growth-delayed E. ictaluri colonies. We also provide safety and efficacy of transposon insertion mutants in catfish. RESULTS: An E. ictaluri transposon insertion library with 45,000 transposants and saturating 30.92% of the TA locations present in the E. ictaluri genome was constructed. Transposon end mapping of 250 growth-delayed E. ictaluri colonies and bioinformatic analysis of sequences revealed 56 unique E. ictaluri genes interrupted by the MAR2xT7 transposon, which are involved in metabolic and cellular processes and mostly localized in the cytoplasm or cytoplasmic membrane. Of the 56 genes, 30 were associated with bacterial virulence. Safety and vaccine efficacy testing of 19 mutants showed that mutants containing transposon insertions in hypothetical protein (Eis::004), and Fe-S cluster assembly protein (IscX, Eis::039), sulfurtransferase (TusA, Eis::158), and universal stress protein A (UspA, Eis::194) were safe and provided significant protection (p < 0.05) against wild-type E. ictaluri. CONCLUSIONS: The results indicate that random transposon mutagenesis causing growth-delayed phenotype results in identification bacterial virulence genes, and attenuated strains with transposon interrupted virulence genes could be used as vaccine to activate fish immune system.

The virulence and immune protection of Edwardsiella ictaluri HemR mutants in catfish.

Edwardsiella ictaluri is a Gram-negative facultative intracellular rod, causing enteric septicemia of catfish (ESC). Several heme uptake systems have been described in bacterial pathogens, most of which involve outer membrane proteins (OMPs). We have shown recently that heme/hemoglobin receptor family protein (HemR) is significantly up-regulated in E. ictaluri under iron-restricted conditions. In this work, our goal was to construct E. ictaluri HemR mutants and assess their virulence and immune protection potentials in catfish. To accomplish this, an in-frame deletion mutant (EiΔhemR) was constructed, and its virulence and immune protection were determined in catfish fingerlings and fry. The results indicated that the EiΔhemR was attenuated completely in catfish fingerlings, but it was virulent in 14 day-old catfish fry. To increase the attenuation of EiΔhemR in fry, we introduced frdA and sdhC gene deletions to the mutant, yielding two double (EiΔhemRΔfrdA and EiΔhemRΔsdhC) and one triple (EiΔhemRΔfrdAΔsdhC) mutants. Results indicated that two double HemR mutants did not exhibit increased attenuation, but the triple HemR mutant showed significantly less virulence and high protection in fry (p < 0.05). Histological examination of fry tissues vaccinated with the triple mutant displayed similar inflammation to that of wild-type infected fry, but much less necrosis and far fewer bacteria were observed. Immunohistochemistry (IHC) result indicated fewer numbers of bacteria around blood vessel and in the hematopoietic tissue in fry infected with triple mutant compared to control group infected with E. ictaluri wild-type. Our data indicated that EiΔhemR was safe and protective in catfish fingerlings, while EiΔhemRΔfrdAΔsdhC was much safer in catfish fry.

Identification of Differentially Regulated Edwardsiella ictaluri Proteins During Catfish Serum Treatment.

Edwardsiella ictaluri is a facultative, intracellular, gram-negative bacterium that causes enteric septicemia of catfish (ESC). Edwardsiella ictaluri is known to be resistant to defense mechanisms present in catfish serum, which might aid in its use of a host's bloodstream to become septicemic. However, the precise mechanisms of the survival of E. ictaluri in host serum are not known. Analysis of the response of E. ictaluri to the host serum treatment at a proteomic level might aid in the elucidation of its adaptation mechanisms against defense mechanisms present in catfish serum. Thus, the objective of this study was to identify differentially regulated proteins of E. ictaluri upon exposure to naïve catfish serum. Two-dimensional difference gel electrophoresis (2D-DIGE) followed by in-gel trypsin digestion and MALDI-TOF/TOF analysis were used for identification of differentially expressed E. ictaluri proteins. A total of 19 differentially regulated proteins (7 up- and 12 downregulated) were identified. Among those were four putative immunogenic proteins, two chaperones and eight proteins involved in the translational process, two nucleic acid degradation and integration proteins, two intermediary metabolism proteins, and one iron-ion-binding protein. Further research focusing on the functions of these differentially expressed proteins may reveal their roles in host adaptation by E. ictaluri.

Salmonella enterica Serovar Kentucky Flagella Are Required for Broiler Skin Adhesion and Caco-2 Cell Invasion.

Nontyphoidal Salmonella strains are the main source of pathogenic bacterial contamination in the poultry industry. Recently, Salmonella enterica serovar Kentucky has been recognized as the most prominent serovar on carcasses in poultry-processing plants. Previous studies showed that flagella are one of the main factors that contribute to bacterial attachment to broiler skin. However, the precise role of flagella and the mechanism of attachment are unknown. There are two different flagellar subunits (fliC and fljB) expressed alternatively in Salmonella enterica serovars using phase variation. Here, by making deletions in genes encoding flagellar structural subunits (flgK, fliC, and fljB), and flagellar motor (motA), we were able to differentiate the role of flagella and their rotary motion in the colonization of broiler skin and cellular attachment. Utilizing a broiler skin assay, we demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables Salmonella motility, but this subunit is unable to mediate tight attachment. Deletion of the flgK gene prevents proper flagellar assembly, making Salmonella significantly less adherent to broiler skin than the wild type. S Kentucky with deletions in all three structural genes, fliC, fljB, and flgK, as well as a flagellar motor mutant (motA), exhibited less adhesion and invasion of Caco-2 cells, while an fljB mutant was as adherent and invasive as the wild-type strain. IMPORTANCE: In this work, we answered clearly the role of flagella in S Kentucky attachment to the chicken skin and Caco-2 cells. We demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables Salmonella motility, but this subunit is unable to mediate strong attachment. Deletion of the flgK gene prevents proper flagellar assembly, making Salmonella significantly less adherent to broiler skin than the wild type. S Kentucky with deletions in all three structural genes, fliC, fljB, and flgK, as well as a flagellar motor mutant (motA), exhibited less adhesion and invasion of Caco-2 cells, while an fljB mutant was as adherent and invasive as the wild-type strain. We expect these results will contribute to the understanding of the mechanisms of Salmonella attachment to food products.

Supplemental invasion of Salmonella from the perspective of Salmonella enterica serovars Kentucky and Typhimurium.

BACKGROUND: Critical to the development of Salmonellosis in humans is the interaction of the bacterium with the epithelial lining of the gastrointestinal tract. Traditional scientific reasoning held type III secretion system (T3SS) as the virulence factor responsible for bacterial invasion. In this study, field-isolated Salmonella enterica serovar Kentucky and a known human pathogen Salmonella enterica serovar Typhimurium were mutated and evaluated for the invasion of human colorectal adenocarcinoma epithelial cells. RESULTS: S. enterica serovar Kentucky was shown to actively invade a eukaryotic monolayer, though at a rate that was significantly lower than Typhimurium. Additionally, strains mutated for T3SS formation were less invasive than the wild-type strains, but the decrease in invasion was not significant in Kentucky. CONCLUSIONS: Strains mutated for T3SS formation were able to initiate invasion of the eukaryotic monolayer to varying degrees based on strain, In the case of Kentucky, the mutated strain initiated invasion at a level that was not significantly different from the wild-type strain. A different result was observed for Typhimurium as the mutation significantly lowered the rate of invasion in comparison to the wild-type strain.

Evaluation of three recombinant outer membrane proteins, OmpA1, Tdr, and TbpA, as potential vaccine antigens against virulent Aeromonas hydrophila infection in channel catfish (Ictalurus punctatus).

A virulent clonal population of Aeromonas hydrophila (VAh) is recognized as the etiological agent in outbreaks of motile aeromonas septicemia (MAS) in catfish aquaculture in the southeastern United States since 2009. Genomic subtraction revealed three outer membrane proteins present in VAh strain ML09-119 but not in low virulence reference A. hydrophila strains: major outer membrane protein OmpA1, TonB-dependent receptor (Tdr), and transferrin-binding protein A (TbpA). Here, the genes encoding ompA1, tdr, and tbpA were cloned from A. hydrophila ML09-119 and expressed in Escherichia coli. The purified recombinant OmpA1, Tdr, and TbpA proteins had estimated molecular weights of 37.26, 78.55, and 41.67 kDa, respectively. Catfish fingerlings vaccinated with OmpA1, Tdr, and TbpA emulsified with non-mineral oil adjuvant were protected against subsequent VAh strain ML09-119 infection with 98.59%, 95.59%, and 47.89% relative percent survival (RPS), respectively. Furthermore, the mean liver, spleen, and anterior kidney bacterial concentrations were significantly lower in catfish vaccinated with the OmpA1 and Tdr than the sham-vaccinated control group. ELISA demonstrated that catfish immunized with OmpA1, Tdr, and TbpA produce significant antibody response by 21 days post-immunization. Therefore, OmpA1 and Tdr proteins could be used as potential candidates for vaccine development against virulent A. hydrophila infection. However, TbpA protein failed to provide strong protection.

Identification of Salmonella enterica serovar Kentucky genes involved in attachment to chicken skin.

BACKGROUND: Regardless of sanitation practices implemented to reduce Salmonella prevalence in poultry processing plants, the problem continues to be an issue. To gain an understanding of the attachment mechanism of Salmonella to broiler skin, a bioluminescent-based mutant screening assay was used. A random mutant library of a field-isolated bioluminescent strain of Salmonella enterica serovar Kentucky was constructed. Mutants' attachment to chicken skin was assessed in 96-well plates containing uniform 6 mm diameter pieces of circular chicken skin. After washing steps, mutants with reduced attachment were selected based on reduced bioluminescence, and transposon insertion sites were identified. RESULTS: Attachment attenuation was detected in transposon mutants with insertion in genes encoding flagella biosynthesis, lipopolysaccharide core biosynthesis protein, tryptophan biosynthesis, amino acid catabolism pathway, shikimate pathway, tricarboxylic acid (TCA) cycle, conjugative transfer system, multidrug resistant protein, and ATP-binding cassette (ABC) transporter system. In particular, mutations in S. Kentucky flagellar biosynthesis genes (flgA, flgC, flgK, flhB, and flgJ) led to the poorest attachment of the bacterium to skin. CONCLUSIONS: The current study indicates that attachment of Salmonella to broiler skin is a multifactorial process, in which flagella play an important role.

Ferric hydroxamate uptake system contributes to Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia in fish, particularly in channel catfish. Ferric iron is an essential micronutrient for bacterial survival, and some bacterial pathogens use secreted hydroxamate-type siderophores to chelate iron in host tissues. Siderophore-iron complexes are taken up by these bacteria via the ferric hydroxamate uptake (Fhu) system. In E. ictaluri, the Fhu system consists of fhuC, fhuD, fhuB, and fhuA genes. However, the importance of the Fhu system in E. ictaluri virulence has not been investigated completely. Here, we present construction of E. ictaluri fhuD and fhuB mutants (EiΔfhuD and EiΔfhuB) by in-frame gene deletion and evaluation of the mutants' virulence and immunogenicity in channel catfish fingerlings and fry. Immersion challenges showed that EiΔfhuD was not significantly attenuated (p < 0.05) in catfish fingerlings, whereas EiΔfhuB was significantly attenuated (p < 0.01). Catfish fingerlings immunized with EiΔfhuD and EiΔfhuB showed 100% and 97.62% survival, respectively. Fry immersion challenges indicated EiΔfhuB was also significantly attenuated (p < 0.05) in two-week old fry compared to the wild-type (48.96% vs. 82.14% mortalities). The survival rate in the fry vaccinated with EiΔfhuB was significantly higher (p < 0.05) than that of non-vaccinated fry (96.77% vs. 21.42% survival). Our data indicates that the fhuB gene, but not the fhuD gene, contributes to E. ictaluri virulence.

Involvement of tolQ and tolR genes in Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative intracellular facultative pathogen causing enteric septicemia of channel catfish (ESC). The Tol system, consisting of four envelope proteins TolQ, TolR, TolA, and TolB, are required for colicin import and contributes to bacterial virulence in several pathogenic bacteria. However, the Tol system and its importance in E. ictaluri virulence have not been investigated. Here we present construction and evaluation of the E. ictaluri TolQ, TolR and TolQR mutants (EiΔtolQ, EiΔtolR, and EiΔtolQR). The Tol mutants were developed using in-frame gene deletion and their attenuation and vaccine efficacy were determined in catfish fingerlings. The EiΔtolQ, EiΔtolR, and EiΔtolQR mutants showed reduced virulence in catfish (28.93%, 19.70%, and 39.82% mortality, respectively) compared to wild type (46.91% mortality). Further, vaccination with these mutants protected catfish against subsequent wild-type infection. This study suggests that the Tol system contributes to E. ictaluri virulence in catfish.

The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication.

Lmof2365_2117 is a Listeria monocytogenes putative cell wall surface anchor protein with a conserved domain found in collagen binding proteins. We constructed a deletion mutation in lmof2365_2117 in serotype 4b strain F2365, evaluated its virulence, and determined its ability to adhere and invade colonic epithelial cells and macrophages. In A/J mice, colonization of liver was significantly higher for F2365 than for F2365Δ2117. The ability of F2365Δ2117 to adhere to Caco-2 cells was significantly lower than F2365. The mutant also showed impaired ability to replicate in intestinal epithelial cell and murine macrophages relative to wild type F2365. Lmof2365_2117 contributed to L. monocytogenes attachment to catfish fillets. Because of its role in adherence to Caco-2 cells, we designated Lmof2365_2117 Listeria adhesion protein B (LapB). The carboxy terminus of LapB is similar to a domain in collagen binding proteins, but our results show that L. monocytogenes does not bind collagen.

Identification of Langerhans-like cells in the immunocompetent tissues of channel catfish, Ictalurus punctatus.

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) that have a critical role in bridging innate and adaptive immune responses in vertebrates. Dendritic cells have been characterized morphologically and functionally in the teleost fish models such as rainbow trout, salmonids, medaka, and zebrafish. The presence of DCs with remarkable similarities to human Langerhans cells (LCs) has been described in the spleen and anterior kidney of salmonids and rainbow trout. However, there is no evidence of the presence of DCs and their role in channel catfish immunity. In this study, we assessed DC-like cells in the immunocompetent tissues of channel catfish by immunohistochemistry (IHC), flow cytometry and transmission electron microscopy (TEM). We identified Langerin/CD207+ (L/CD207+) cells in the channel catfish anterior kidney, spleen and gill by IHC. Moreover, we described the cells that resembled mammal LC DCs containing Birbeck-like (BL) granules in channel catfish spleen, anterior and posterior kidneys and gill by TEM. Our data suggest that cells with DC-like morphology in the immune related organs of catfish may share morphological and functional properties with previously reported DCs in teleost fish and mammals. More detailed knowledge of the phenotype and the function of catfish DCs will not only help gain insight into the evolution of the vertebrate adaptive immune system but will also provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use.

A novel suicide plasmid for efficient gene mutation in Listeria monocytogenes.

Although several plasmids have been used in Listeria monocytogenes for generating mutants by allelic exchange, construction of L. monocytogenes mutants has been inefficient due to lack of effective selection markers for first and second recombination events. To address this problem, we have developed a new suicide plasmid, pHoss1, by using the pMAD plasmid backbone and anhydrotetracycline selection marker (secY antisense RNA) driven by an inducible Pxyl/tetO promoter. Expression of the secY antisense RNA eliminates merodiploids and selects for the loss of plasmid via a second allelic exchange, which enriches the number of mutants with deleted genes. To assess the effectiveness of pHoss1 for the generation of stable in-frame deletion mutations, we deleted the ispG and ispH genes of L. monocytogenes serotype 4b strain F2365. Results showed that identification of the second allelic exchange mutants was very efficient with 80-100% of the colonies yielding desired deletion mutants. L. monocytogenes' intestinal cell attachment was not altered when ispG and ispH genes were deleted. We expect that this new plasmid will be very useful for construction of marker-free deletion mutants in L. monocytogenes and in other Gram-positive bacteria, including Staphylococcus aureus and Bacillus cereus.

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 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.