Small RNA molecules are short, non-coding RNAs identified for their crucial role in post-transcriptional regulation. A well-studied example includes miRNAs (microRNAs) which have been identified in several model organisms including the freshwater flea and planktonic crustacean Daphnia. A model for epigenetic-based studies with an available genome database, the identification of miRNAs and their potential role in regulating Daphnia gene expression has only recently garnered interest. Computational-based work using Daphnia pulex, has indicated the existence of 45 miRNAs, 14 of which have been experimentally verified. To extend this study, we took a sequencing approach towards identifying miRNAs present in a small RNA library isolated from Daphnia magna. Using Perl codes designed for comparative genomic analysis, 815,699 reads were obtained from 4 million raw reads and run against a database file of known miRNA sequences. Using this approach, we have identified 205 putative mature miRNA sequences belonging to 188 distinct miRNA families. Data from this study provides critical information necessary to begin an investigation into a role for these transcripts in the epigenetic regulation of Daphnia magna.

Edwardsiella ictaluri is a Gram-negative facultative anaerobic rod and the causative agent of enteric septicemia of channel catfish (ESC), which is one of the most prevalent diseases of catfish, causing significant economic losses in the catfish industry. E. ictaluri is resistant to complement system and macrophage killing, which results in rapid systemic septicemia. However, mechanisms of E. ictaluri stress responses under conditions of host environment are not studied well. Therefore, in this work, we report E. ictaluri stress responses during hydrogen peroxide, low pH, and catfish serum stresses as well as during catfish invasion. E. ictaluri stress responses were characterized by identifying expression of 13 universal stress protein (USP) genes (usp01-usp13) and seven USP-interacting protein genes (groEL, groES, dnaK, grpE, and clpB, grpE, relA). Data indicated that three usp genes (usp05, usp07, and usp13) were highly expressed in all stress conditions. Similarly, E. ictaluri heat shock proteins groEL, groES, dnaK, grpE, and clpB were highly expressed in oxidative stress. Also, E. ictaluri grpE and relA were highly expressed in catfish spleen and head kidney. These findings contribute to our understanding of stress response mechanisms in E. ictaluri stress response, and stress-related proteins that are essential for E. ictaluri could be potential targets for live attenuated vaccine development against ESC.

Edwardsiella ictaluri is a Gram-negative intracellular pathogen causing enteric septicemia of channel catfish (ESC). Type six secretion system (T6SS) is a sophisticated nanomachine that delivers effector proteins into eukaryotic host cells as well as other bacteria. In the current work, we in-frame deleted the E. ictalurievpB gene located in the T6SS operon by allelic exchange. The safety and efficacy of EiΔevpB as well as Aquavac-ESC, a commercial vaccine manufactured by Intervet/Merck Animal Health, were evaluated in channel catfish (Ictalurus punctatus) fingerlings and fry by immersion exposure. Our results showed that the EiΔevpB strain was avirulent and fully protective in catfish fingerlings. The EiΔevpB strain was also safe in catfish fry, and immersion vaccination with EiΔevpB at doses 106 and 107 CFU/ml in water resulted in 34.24 and 80.34% survival after wild-type immersion challenge compared to sham-vaccinated fry (1.79% survival). Catfish fry vaccinated with EiΔevpB at doses 106, 107, and 108 CFU/ml in water exhibited dose-dependent protection. When compared with Aquavac-ESC, EiΔevpB provided significantly higher protection in catfish fingerlings and fry (p < 0.05). Results indicate that the EiΔevpB strain is safe and can be used to protect catfish fingerlings and fry against E. ictaluri.

Edwardsiella tarda is a Gram-negative facultative anaerobe causing disease in animals and humans. Here, we announce the complete genome sequence of the channel catfish isolate E. tarda strain C07-87, which was isolated from an outbreak of gastrointestinal septicemia on a commercial catfish farm.

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.

Columnaris disease caused by Gram-negative rod Flavobacterium columnare is one of the most common diseases of catfish. F. columnare is also a common problem in other cultured fish species worldwide. F. columnare has three major genomovars; we have sequenced a representative strain from genomovar I (ATCC 49512, which is avirulent in catfish) and genomovar II (94-081, which is highly pathogenic in catfish). Here, we present a comparative analysis of the two genomes. Interestingly, F. columnare ATCC 49512 and 94-081 meet criteria to be considered different species based on the Average Nucleotide Identity (90.71% similar) and DNA-DNA Hybridization (42.6% similar). Genome alignment indicated the two genomes have a large number of rearrangements. However, function-based comparative genomics analysis indicated that the two strains have similar functional capabilities with 2,263 conserved orthologous clusters; strain ATCC 49512 has 290 unique orthologous clusters while strain 94-081 has 391. Both strains carry type I secretion system, type VI secretion system, and type IX secretion system. The two genomes also have similar CRISPR capacities. The F. columnare strain ATCC 49512 genome contains a higher number of insertion sequence families and phage regions, while the F. columnare strain 94-081 genome has more genomic islands and more regulatory gene capacity. Transposon mutagenesis using Tn4351 in pathogenic strain 94-081 yielded six mutants, and experimental infections of fish showed hemolysin and glycine cleavage protein mutants had 15 and 10% mortalities, respectively, while the wild-type strain caused 100% mortalities. Our comparative and mutational analysis yielded important information on classification of genomovars I and II F. columnare as well as potential virulence genes in F. columnare strain 94-081.

A highly virulent clonal population of Aeromonas hydrophila (vAh) has been the cause of recent motile Aeromonas septicemia epizootic in channel catfish (Ictalurus punctatus) farms in the Southeastern United States. The pathology of the disease caused by vAh has not been studied well yet. Thus, our aim was to determine histopathological and ultrastructural changes in channel catfish following vAh challenge. To accomplish this, catfish fingerlings were challenged with vAh (strain ML09-119) by bath. Six fish per each time point were collected at 1, 3, 5, 6, 24, and 48 h for light microscopy, and six fish were collected at 48 h for transmission electron microscopy (TEM). The first pathological lesions were detected in the spleen and stomach at 1 h post-challenge (HPC) while intestine, gills, kidney, and liver lesions were observed at 24 and 48 HPC. Histopathological examination revealed degenerative changes, necrosis, extensive edema, and inflammation in internal organs. The TEM showed severe tissue destruction with multiple bacterial cells secreting outer membrane vesicles, especially in spleen and gills and far number in the stomach. Degenerated bacterial cells were observed in the intestinal lumen and the phagosomes of phagocytic kidney cells. We identified, for the first time, degranulate eosinophilic granular cells, and dendritic cells like (DC-like) cells in the necrotic intestinal epithelium. These findings suggest that vAh rapidly proliferated and spread through the catfish organs following bath challenge.

Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed 'pJAZZ' vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT-rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries.

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.

Cyclosporine is a potent immunosuppressive agent used to treat immune-mediated disorders in dogs. Secondary infections sometimes necessitate withdrawal of cyclosporine, but it is not known how long it takes for the immune system to recover after cessation of cyclosporine. Our goal was to utilize a validated RT-qPCR assay in dogs to assess recovery time of the T-cell cytokines IL-2 and IFN-γ after discontinuation of cyclosporine. Six healthy dogs were given oral cyclosporine (10 mg/kg every 12 hr) for 1 week, with samples collected for measurement of cytokine gene expression prior to treatment, and on the last day of therapy. Cyclosporine was then discontinued, and samples were collected daily for an additional 7 days. Results revealed that there was a significant difference in cytokine expression when comparing pre-treatment and immediate post-treatment values, corresponding to marked suppression of T-cell function. There was no significant difference between pre-treatment values for either cytokine when compared with any day during the recovery period. Cytokine expression, evaluated as a percentage of pre-treatment baseline samples, demonstrated progressing return of T-cell function after drug cessation, with full recovery seen in all dogs by Day 4 of the recovery period.

Listeria monocytogenes is a ubiquitous opportunistic foodborne pathogen capable of survival in various adverse environmental conditions. Pathogenesis of L. monocytogenes is tightly controlled by a complex regulatory network of transcriptional regulators that are necessary for survival and adaptations to harsh environmental conditions both inside and outside host cells. Among these regulatory pathways are members of the DeoR-family transcriptional regulators that are known to play a regulatory role in sugar metabolism. In this study, we deciphered the role of FruR, a DeoR family protein, which is a fructose operon transcriptional repressor protein, in L. monocytogenes pathogenesis and growth. Following intravenous (IV) inoculation in mice, a mutant strain with deletion of fruR exhibited a significant reduction in bacterial burden in liver and spleen tissues compared to the parent strain. Further, the ΔfruR strain had a defect in cell-to-cell spread in L2 fibroblast monolayers. Constitutive activation of PrfA, a pleiotropic activator of L. monocytogenes virulence factors, did not restore virulence to the ΔfruR strain, suggesting that the attenuation was not a result of impaired PrfA activation. Transcriptome analysis revealed that FruR functions as a positive regulator for genes encoding enzymes involved in the pentose phosphate pathway (PPP) and as a repressor for genes encoding enzymes in the glycolysis pathway. These results suggested that FruR may function to facilitate NADPH regeneration, which is necessary for full protection from oxidative stress. Interestingly, deletion of fruR increased sensitivity of L. monocytogenes to H2O2, confirming a role for FruR in survival of L. monocytogenes during oxidative stress. Using anti-mouse neutrophil/monocyte monoclonal antibody RB6-8C5 (RB6) in an in vivo infection model, we found that FruR has a specific function in protecting L. monocytogenes from neutrophil/monocyte-mediated killing. Overall, this work clarifies the role of FruR in controlling L. monocytogenes carbon flow between glycolysis and PPP for NADPH homeostasis, which provides a new mechanism allowing metabolic adaptation of L. monocytogenes to oxidative stress.

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.

Flavobacterium columnare causes columnaris disease in cultured and wild fish populations worldwide. Columnaris is the second most prevalent bacterial disease of commercial channel catfish industry in the United States. Despite its economic importance, little is known about the expressed proteins and virulence mechanisms of F. columnare. Here, we report the first high throughput proteomic analysis of F. columnare using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.

Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE) for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS) for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05) difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri.

Listeria monocytogenes is an intracellular facultative pathogen that causes listeriosis, a foodborne zoonotic infection. There are differences in the pathogenic potential of L. monocytogenes subtypes and strains. Comparison of the genome sequences among L. monocytogenes pathogenic strains EGD-e and F2365 with nonpathogenic L. innocua CLIP1182 and L. monocytogenes strain HCC23 revealed a set of proteins that were present in pathogenic strains and had no orthologs among the nonpathogenic strains. Among the candidate virulence factors are five proteins: putrescine carbamoyltransferase; InlH/InlC2 family class 1 internalin; phosphotransferase system (PTS) fructose transporter subunit EIIC; putative transketolase; and transcription antiterminator BglG family. To determine if these proteins have a role in adherence and invasion of intestinal epithelial Caco-2 cells and/or contribute to virulence, five mutant strains were constructed. F2365ΔinlC2, F2365Δeiic, and F2365Δtkt exhibited a significant (p < 0.05) reduction in adhesion to Caco-2 cells compared to parent F2365 strain. The invasion of F2365ΔaguB, F2365ΔinlC2, and F2365ΔbglG decreased significantly (p < 0.05) compared with the parent strain. Bacterial loads in mouse liver and spleen infected by F2365 was significantly (p < 0.05) higher than it was for F2365ΔaguB, F2365ΔinlC2, F2365Δeiic, F2365Δtkt, and F2365ΔbglG strains. This study demonstrates that aguB, inlC2, eiic, tkt, and bglG play a role in L. monocytogenes pathogenicity.

Edwardsiella ictaluri is an intracellular Gram-negative facultative pathogen causing enteric septicemia of catfish (ESC), a common disease resulting in substantial economic losses in the U.S. catfish industry. Previously, we demonstrated that several universal stress proteins (USPs) are highly expressed under in vitro and in vivo stress conditions, indicating their importance for E. ictaluri survival. However, the roles of these USPs in E. ictaluri virulence is not known yet. In this work, 10 usp genes of E. ictaluri were in-frame deleted and characterized in vitro and in vivo. Results show that all USP mutants were sensitive to acidic condition (pH 5.5), and EiΔusp05 and EiΔusp08 were very sensitive to oxidative stress (0.1% H2O2). Virulence studies indicated that EiΔusp05, EiΔusp07, EiΔusp08, EiΔusp09, EiΔusp10, and EiΔusp13 were attenuated significantly compared to E. ictaluri wild-type (EiWT; 20, 45, 20, 20, 55, and 10% vs. 74.1% mortality, respectively). Efficacy experiments showed that vaccination of catfish fingerlings with EiΔusp05, EiΔusp07, EiΔusp08, EiΔusp09, EiΔusp10, and EiΔusp13 provided complete protection against EiWT compared to sham-vaccinated fish (0% vs. 58.33% mortality). Our results support that USPs contribute E. ictaluri virulence in catfish.

Virulent Aeromonas hydrophila causes severe motile Aeromonas septicemia in warmwater fishes. In recent years, channel catfish farming in the U.S.A. and carp farming in China have been affected by virulent A. hydrophila, and genome comparisons revealed that these virulent A. hydrophila strains belong to the same clonal group. Bacterial secretion systems are often important virulence factors; in the current study, we investigated whether secretion systems contribute to the virulent phenotype of these strains. Thus, we conducted comparative secretion system analysis using 55 A. hydrophila genomes, including virulent A. hydrophila strains from U.S.A. and China. Interestingly, tight adherence (TaD) system is consistently encoded in all the vAh strains. The majority of U.S.A. isolates do not possess a complete type VI secretion system, but three core elements [tssD (hcp), tssH, and tssI (vgrG)] are encoded. On the other hand, Chinese isolates have a complete type VI secretion system operon. None of the virulent A. hydrophila isolates have a type III secretion system. Deletion of two genes encoding type VI secretion system proteins (hcp1 and vgrG1) from virulent A. hydrophila isolate ML09-119 reduced virulence 2.24-fold in catfish fingerlings compared to the parent strain ML09-119. By determining the distribution of genes encoding secretion systems in A. hydrophila strains, our study clarifies which systems may contribute to core A. hydrophila functions and which may contribute to more specialized adaptations such as virulence. Our study also clarifies the role of type VI secretion system in A. hydrophila virulence.

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.

Edwardsiella ictaluri causes enteric septicemia of catfish. Our group developed two E. ictaluri live attenuated vaccines (LAVs). However, their effects on the innate functions of catfish B cells are still unexplored. We evaluated phagocytosis and killing of wild-type (WT) E. ictaluri opsonized with sera from vaccinated fish and the survival of B cells exposed to E. ictaluri strains. We assessed phagocytosis of the opsonized WT at 30 °C and 4 °C. B cells killed the internalized E. ictaluri opsonized with sera from vaccinated fish with LAVs more efficiently than other groups at 30 °C. However, catfish B cells were unable to destroy E. ictaluri at 4 °C. Furthermore, E. ictaluri opsonized with serum from fish exposed to WT induce apoptosis and decreased live B cells numbers. Results indicate that opsonization of E. ictaluri with sera from vaccinated fish enhanced phagocytosis and killing activity in B cells and inhibited apoptotic changes in the infected B cells.

One molecular-based approach that increases potency and reduces dose-limited sequela is the implementation of selective 'targeted' delivery strategies for conventional small molecular weight chemotherapeutic agents. Descriptions of the molecular design and organic chemistry reactions that are applicable for synthesis of covalent gemcitabine-monophosphate immunochemotherapeutics have to date not been reported. The covalent immunopharmaceutical, gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R] was synthesized by reacting gemcitabine with a carbodiimide reagent to form a gemcitabine carbodiimide phosphate ester intermediate which was subsequently reacted with imidazole to create amine-reactive gemcitabine-(5'-phosphorylimidazolide) intermediate. Monoclonal anti-IGF-1R immunoglobulin was combined with gemcitabine-(5'-phosphorylimidazolide) resulting in the synthetic formation of gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R]. The gemcitabine molar incorporation index for gemcitabine-(5'-phosphoramidate)-[anti-IGF-R1] was 2.67:1. Cytotoxicity Analysis - dramatic increases in antineoplastic cytotoxicity were observed at and between the gemcitabine-equivalent concentrations of 10-9  M and 10-7  M where lethal cancer cell death increased from 0.0% to a 93.1% maximum (100.% to 6.93% residual survival), respectively. Advantages of the organic chemistry reactions in the multistage synthesis scheme for gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R] include their capacity to achieve high chemotherapeutic molar incorporation ratios; option of producing an amine-reactive chemotherapeutic intermediate that can be preserved for future synthesis applications; and non-dedicated organic chemistry reaction scheme that allows substitutions of either or both therapeutic moieties, and molecular delivery platforms.