Potencijalna upotreba auksotrofnih aroa i aroc mutanata yersinia ruckeri kao žive atenuirane vakcine

Yersinia ruckeri causes yersiniosis in rainbow trout (Oncorhynchus mykiss). Yersiniosis is the main causes of high mortalities and severe economic losses in freshwater and marine aquaculture. To treat and prevent yersiniosis, antibiotics and inactive vaccines have been used. However, use of antibiotics can cause antibiotic resistance in bacteria, while inactive vaccines do not provide prolonged protection against bacterial fish diseases. Formation of antibiotic resistance in bacteria against antibiotics, and chemical contamination of the environment are some of the undesirable outcomes. For these reasons, prevention of fish diseases using vaccination strategies is important for ensuring the profitability and sustainability of aquaculture production. The objective of this research is to develop live attenuated vaccines against yersiniosis. To reach this aim, aroA and aroC genes of Y. ruckeri have been mutated and virulence and efficacy of these mutants are characterized. Our main hypothesis is that Y. ruckeri with mutations in their aromatic amino acid biosynthesis network (aro) will lose their ability to cause infections in fish and these will be used as live vaccines. To accomplish the aim of this research, 5' and 3' regions of Y. ruckeri aroA (5-enolpyruvylshikimate-3-phosphate syntheses) and aroC (2, 3-dihydroxybenzoic acid) genes are amplified and DNA fragments mutated by overlap extension PCR will be cloned into a suicide plasmid (pDS132). This plasmid will be transferred to Y. ruckeri for replacing wild type genes with mutated aroA and aroC genes via homologous recombination. Successful completion of this phase is expected to yield live attenuated vaccine candidates. It is expected that these live attenuated vaccines will provide resistance against the wild type Y. ruckeri infections in trout, and thus, prevent onset and progress of diseases. Successful completion of this study is expected to prevent fish losses due to Y. ruckeri infections and increase the profitability of aquaculture. Contend with fish diseases using traditional methods is generally ineffective and expensive

Proteomic analysis of the fish pathogen Flavobacterium columnare

<p>Abstract</p> <p>Background</p> <p><it>Flavobacterium columnare </it>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 <it>F. columnare</it>. Here, we report the first high throughput proteomic analysis of <it>F. columnare </it>using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.</p> <p>Results</p> <p>Proteins identified in this study and predicted from the draft <it>F. columnare </it>genome were clustered into functional groups using clusters of orthologous groups (COGs), and their subcellular locations were predicted. Possible functional relations among the identified proteins were determined using pathway analysis. The total number of unique <it>F. columnare </it>proteins identified using both 2-D LC and 2-DE approaches was 621, of which 10.95% (68) were identified by both methods, while 77.29% (480) and 11.76% (73) were unique in 2-D LC and 2-DE, respectively. COG groupings and subcellular localizations were similar between our data set and proteins predicted from the whole genome. Twenty eight pathways were significantly represented in our dataset (<it>P </it>< 0.05).</p> <p>Conclusion</p> <p>Results from this study provide experimental evidence for many proteins that were predicted from the <it>F. columnare </it>genome annotation, and they should accelerate functional and comparative studies aimed at understanding virulence mechanisms of this important pathogen.</p

Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon

<p>Abstract</p> <p>Background</p> <p>Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent <it>Salmonella enterica </it>serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of <it>luxCDABE </it>genes into the chromosome of eleven <it>Salmonella enterica </it>serotypes isolated from the broiler production continuum.</p> <p>Results</p> <p>We found that the <it>lux </it>operon is constitutively expressed from the chromosome post-transposition and the <it>lux </it>cassette is stable without external pressure, i.e. antibiotic selection, for all <it>Salmonella enterica </it>serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies.</p> <p>Conclusions</p> <p>This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for <it>Salmonella </it>on food products in real-time for both short and long term studies.</p

Comparative Genomics of Aeromonas hydrophila Secretion Systems and Mutational Analysis of hcp1 and vgrG1 Genes From T6SS

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

Assessment of the Live Attenuated and Wild-Type Edwardsiella ictaluri-Induced Immune Gene Expression and Langerhans-Like Cell Profiles in the Immune-Related Organs of Catfish

Edwardsiella ictaluri is a Gram-negative intracellular pathogen that causes enteric septicemia of catfish (ESC). Successful vaccination against intracellular pathogens requires T cell priming by antigen presenting cells (APCs) that bridge innate and adaptive immunity. However, the evidence on immunological mechanisms that underscore E. ictaluri pathogenesis and the protective role of live attenuated vaccines (LAVs) is scarce. We assessed the expression of immune genes related to antigen presentation by real-time PCR and the distribution patterns of Langerhans-like (L/CD207+) cells by immunohistochemistry in the immune-related tissues of channel catfish challenged with two novel E. ictaluri LAVs, EiΔevpB, and ESC-NDKL1 and wild type (WT) strain. Our results indicated significantly elevated expression of IFN-γ gene in the anterior kidney (AK) and spleen of vaccinated catfish at the early stages of exposure, which correlated with increased numbers of L/CD207+ cells. In general, the ESC-NDKL1-induced IFN-γ gene expression patterns in the AK resembled that of the patterns induced by EiΔevpB. However the MHCII gene expression patterns differed between the strains with significant increases at 6 h post-challenge (pc) with the EiΔevpB and at 7 d pc with the ESC-NDKL1 strains, respectively. Significant increases in activity of T helper type polarization genes such as IFN-γ and T cell co-receptors after exposure to ESC-NDKL1, in combination with elevated numbers of L/CD207+ cells at 7 d pc with both LAVs compared to uninfected and the WT-exposed counterparts, were documented in the spleen. The dominant pro-inflammatory environment with dramatically overexpressed inflammatory genes in the AK and 7 d pc in the spleen in response to E. ictaluri was found in exposed catfish. In general, the pro-inflammatory gene expression profiles in the ESC-NDKL1 pc showed more similarities to the WT strain-induced gene profiles compared to the EiΔevpB counterpart. In addition, E. ictaluri WT significantly decreased the numbers of Langerhans-like L/CD207+ cells in the AK and spleen at 3 and 7 days pc. In conclusion, we report the differential framework of initiation of innate and adaptive immune responses between E. ictaluri strains with both LAVs having a potential of satisfying the stringent requirements for successful vaccines

Universal Stress Proteins Contribute Edwardsiella ictaluri Virulence in Catfish

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

Taxonomic and Functional Metagenomic Profile of Sediment From a Commercial Catfish Pond in Mississippi

Metagenomic analyses of microbial communities from aquatic sediments are relatively few, and there are no reported metagenomic studies on sediment from inland ponds used for aquaculture. Catfish ponds in the southeastern U.S. are eutrophic systems. They are fertilized to enhance algae growth and encourage natural food production, and catfish are fed with commercial feed from spring to fall. As result, catfish pond sediment (CPS) contains a very dense, diverse microbial community that has significant effects on the physiochemical parameters of pond dynamics. Here we conducted an in-depth metagenomic analysis of the taxonomic and metabolic capabilities of a catfish pond sediment microbiome from a southeastern U.S. aquaculture farm in Mississippi using Illumina next-generation sequencing. A total of 3.3 Gbp of sequence was obtained, 25,491,518 of which encoded predicted protein features. The pond sediment was dominated by Proteobacteria sequences, followed by Bacteroidetes, Firmicutes, Chloroflexi, and Actinobacteria. Enzyme pathways for methane metabolism/methanogenesis, denitrification, and sulfate reduction appeared nearly complete in the pond sediment metagenome profile. In particular, a large number of Deltaproteobacteria sequences and genes encoding anaerobic functional enzymes were found. This is the first study to characterize a catfish pond sediment microbiome, and it is expected to be useful for characterizing specific changes in microbial flora in response to production practices. It will also provide insight into the taxonomic diversity and metabolic capabilities of microbial communities in aquaculture. Furthermore, comparison with other environments (i.e., river and marine sediments) will reveal habitat-specific characteristics and adaptations caused by differences in nutrients, vegetation, and environmental stresses

Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli

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