A recombinant 9E1 monoclonal antibody binds membrane and soluble channel catfish immunoglobulin M.

The development and validation of the recombinant 9E1 monoclonal antibody against channel catfish IgM is described. The variable heavy and light chain domains of the 9E1 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into 293F cells and mature IgG was purified from culture supernatant. It is demonstrated that the recombinant 9E1 monoclonal antibody binds to soluble IgM in ELISA and ELISPOT assays and to membrane-bound IgM by immunofluorescence with different B-cell types. The recombinant 9E1 monoclonal antibody will be a valuable tool in the continued examination of the channel catfish adaptive immune system.

Whole-Genome Sequencing and Annotation of Aeromonas veronii Isolates from Channel Catfish.

The genomes of seven Aeromonas veronii strains isolated from tissues of healthy or diseased channel catfish obtained from Alabama, USA, fish farms were sequenced and annotated. These genome sequences will enable comparative analyses to determine the roles these bacteria play in catfish aquaculture and the development of new preventative or management strategies.

Transcriptome analysis of Pacific white shrimp (Liptopenaeus vannamei) after exposure to recombinant Vibrio parahaemolyticus PirA and PirB proteins.

Vibrio parahaemolyticus is a Gram-negative bacterium commonly found in marine and estuarine environments and is endemic among the global shrimp aquaculture industry. V. parahaemolyticus proteins PirA and PirB have been determined to be major virulence factors that contribute significantly to the development of acute hepatopancreatic necrosis disease. Our previous work had demonstrated the lethality of recombinant PirA and PirB proteins to Pacific white shrimp (Liptopenaeus vannamei). To understand the host response to these proteins, recombinant PirA and PirB proteins were administered using a reverse gavage method and individual shrimp were then sampled over time. Shrimp hepatopancreas libraries were generated and RNA sequencing was performed on the control and recombinant PirA/B-treated samples. Differentially expressed genes were identified among the assayed time points. Differentially expressed genes that were co-expressed at the later time points (2-, 4- and 6-h) were also identified and gene associations were established to predict functional physiological networks. Our analysis reveals that the recombinant PirA and PirB proteins have likely initiated an early host response involving several cell survival signaling and innate immune processes.

Transcriptome analysis and immune gene expression of channel catfish (Ictalurus punctatus) fed diets with inclusion of frass from black soldier fly larvae.

The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. In this study, we subjected channel catfish (Ictalurus punctatus) to a 10-week feeding trial to assess how different dietary amounts of frass inclusion would affect both systemic and mucosal tissue gene expression, especially in regard to growth and immune-related genes. Fish were divided in quadruplicate aquaria, and five experimental diets comprising 0, 50, 100, 200, and 300 g of frass per kilogram of feed were fed twice daily. At the end of the trial, liver, head kidney, gill, and intestine samples were collected for gene expression analyses. First, liver and intestine samples from fish fed with a no frass inclusion diet (control), low-frass (50 g/kg) inclusion diet, or a high-frass (300 g/kg) inclusion diet were subjected to Illumina RNA sequencing to determine global differential gene expression among diet groups. Differentially expressed genes (DEGs) included the upregulation of growth-related genes such as glucose-6-phosphatase and myostatin, as well as innate immune receptors and effector molecules such as toll-like receptor 5, apolipoprotein A1, C-type lectin, and lysozyme. Based on the initial screenings of low/high frass using RNA sequencing, a more thorough evaluation of immune gene expression of all tissues sampled, and all levels of frass inclusion, was further conducted. Using targeted quantitative PCR panels for both innate and adaptive immune genes from channel catfish, differential expression of genes was identified, which included innate receptors (TLR1, TLR5, TLR9, and TLR20A), proinflammatory cytokines (IL-1ß type a, IL-1ß type b, IL-17, IFN-γ, and TNFα), chemokines (CFC3 and CFD), and hepcidin in both systemic (liver and head kidney) and mucosal (gill and intestine) tissues. Overall, frass from black soldier fly larvae inclusion in formulated diets was found to alter global gene expression and activate innate and adaptive immunity in channel catfish, which has the potential to support disease resistance in this species in addition to demonstrated growth benefits.

Draft Genome Sequences of Flavobacterium covae Strains LSU-066-04 and LV-359-01.

Flavobacterium covae is one of four Flavobacterium spp. that cause columnaris disease in teleost fish. Here, we report the draft genomes of two isolates, LSU-066-04 and LV-359-01, and their predicted virulence factors.

The effect of piscidin antimicrobial peptides on the formation of Gram-negative bacterial biofilms.

Fish-derived antimicrobial peptides are an important part of the innate immune system due to their potent antimicrobial properties. Piscidins are a class of antimicrobial peptides first described in hybrid striped bass (Morone chrysops x Morone saxatilis) but have also been identified in many other fish species. Previous work demonstrated the broad antimicrobial activity of piscidins against Gram-negative and Gram-positive bacterial species. This study sought to determine the extent to which class I (striped bass piscidin 1, white bass piscidin 1 and striped bass/white bass piscidin 3) and class II (striped bass piscidin 4 and white bass piscidin 5) piscidins inhibit biofilm formation of different Gram-negative bacteria. In general, the class I and II piscidins demonstrate potent activity against Escherichia coli and Flavobacterium columnare biofilms. The class II piscidins showed more activity against E. coli and F. columnare isolates than did the class I piscidins. The piscidins in general were much less effective against inhibiting Aeromonas hydrophila and A. veronii biofilm growth. Only the class I piscidins showed significant growth inhibition among the Aeromonas spp. examined.

Toxicity of recombinant PirA and PirB derived from Vibrio parahaemolyticus in shrimp.

Acute hepatopancreatic necrosis disease (AHPND), caused by emerging strains of Vibrio Parahaemolyticus, is of concern in shrimp aquaculture. Secreted proteins PirA and PirB, encoded by a plasmid harbored in V. parahaemolyticus, were determined to be the major virulence factors that induce AHPND. To better understand pathogenesis associated with PirA and PirB, recombinant proteins rPirA and rPirB were produced to evaluate their relative toxicities in shrimp. By challenging shrimp at concentration of 3 µM with reverse gavage method, rPirA and rPirB (approximately 0.4 and 1.5 µg per g of body weight, respectively) caused 27.8 ± 7.8% and 33.3 ± 13.6% mortality, respectively; combination of 3 µM rPirA and rPirB resulted in 88.9 ± 7.9% mortality. Analysis of protein mobility in native gel revealed that rPirB was apparently in the form of monomer while rPirA was oligomerized as an octamer-like macromolecule, suggesting that inter- and intra-molecular interactions between rPirA and rPirB enhanced the toxic effect. An attempt to block or reduce rPirA activity with a putative receptor, N-acetyl-galactosamine, was unsuccessful, implying that remodeling analysis of PirA molecule, such as the octamer observed in this study, is necessary. Results of this study provided new insight into toxic mechanism of PirA and PirB and shall help design strategic antitoxin methods against AHPND in shrimp.

Differential susceptibility of white bass (Morone chrysops), striped bass (Morone saxatilis) and hybrid striped bass (M. chrysops × M. saxatilis) to Flavobacterium columnare and effects of mucus on bacterial growth and biofilm development.

Columnaris disease generates substantial losses of many freshwater fish species; one is the hybrid striped bass. The ubiquitous aquatic bacterium Flavobacterium columnare can be highly effective in biofilm formation on fish skin and gills. Previous research showed a difference between columnaris disease susceptibility of hybrid striped bass (Morone saxatilis × M. chrysops) and white bass (M. chrysops). To understand these differential susceptibilities and possible mucosal relationship, we assessed total bacterial growth and biofilm formation with mucus derived from each moronid parental species: white bass and striped bass (M. saxatilis). Differential susceptibility was confirmed of the other parent species, the striped bass (M. saxatilis). In addition to intraspecies investigations, individual hybrid striped bass mucosal affects were also studied for deferential responses to bacterial growth and biofilm formation. Species- and concentration-dependent differences were detected in the total growth of the bacteria to host mucus. Our data suggest that bass mucus can significantly affect biofilm formation with the F. columnare isolate tested. There appears to be a correlation between the bacteria's response of growth and biofilms and bass species susceptibility. This study provides insight into our understanding of the host-pathogen interaction between F. columnare and moronids.

Proteome analysis of virulent Aeromonas hydrophila reveals the upregulation of iron acquisition systems in the presence of a xenosiderophore.

The Gram-negative bacterium, Aeromonas hydrophila, has been responsible for extensive losses in the catfish industry for over a decade. Due to this impact, there are ongoing efforts to understand the basic mechanisms that contribute to virulent A. hydrophila (vAh) outbreaks. Recent challenge models demonstrated that vAh cultured in the presence of the iron chelating agent deferoxamine mesylate (DFO) were more virulent to channel catfish (Ictalurus punctatus). Interestingly, differential gene expression of select iron acquisition genes was unremarkable between DFO and non-DFO cultures, posing the question: why the increased virulence? The current work sought to evaluate growth characteristics and protein expression of vAh after the addition of DFO. A comparative proteome analysis revealed differentially expressed proteins among tryptic soy broth (TSB) and TSB + DFO treatments. Upregulated proteins identified among the TSB + DFO treatment were enriched for gene ontology groups including iron ion transport, siderophore transport and siderophore uptake transport, all iron acquisition pathways. Protein-protein interactions were also evaluated among the differentially expressed proteins and predicted that many of the upregulated iron acquisition proteins likely form functional physiological networks. The proteome analysis of the vAh reveals valuable information about the basic biological processes likely leading to increased virulence during iron restriction in this organism.

Vertebrate mucus stimulates biofilm development and upregulates iron acquisition genes in Flavobacterium columnare.

Columnaris disease is responsible for substantial losses throughout the production of many freshwater fish species. One of the ways in which the bacterium Flavobacterium columnare is so effective in initiating disease is through the formation of biofilms on fish skin and gills. To further explore the interaction between host factors and bacterial cells, we assayed the ability of vertebrate mucus to enhance F. columnare biofilm development. Different concentrations of catfish, tilapia and pig mucus (5-60 µg/ml) increased biofilm growth at varying degrees among F. columnare isolates. Our data suggest that vertebrate mucus acts as a signalling molecule for the development of F. columnare biofilms; however, there are clear disparities in how individual isolates respond to different mucus fractions to stimulate biofilms. The expression of iron acquisition genes among two genomovar II isolates showed that ferroxidase, TonB receptor and the siderophore synthetase gene were all significantly upregulated among F. columnare biofilms. Interestingly, the siderophore acetyltransferase gene was only shown to be significantly upregulated in one of the genomovar II isolates. This work provides insight into our understanding of the interaction between F. columnare and vertebrate mucus, which likely contributes to the growth of planktonic cells and the transition into biofilms.

Evaluation of a Recombinant Flavobacterium columnare DnaK Protein Vaccine as a Means of Protection Against Columnaris Disease in Channel Catfish (Ictalurus punctatus).

Flavobacterium columnare causes substantial losses among cultured finfish species. The Gram-negative bacterium is an opportunistic pathogen that manifests as biofilms on the host's mucosal surfaces as the disease progresses. We previously demonstrated that the dominant mucosal IgM antibody response to F. columnare is to the chaperone protein DnaK that is found in the extracellular fraction. To establish the efficacy of using recombinant protein technology to develop a new vaccine against columnaris disease, we are reporting on two consecutive years of vaccine trials using a recombinant F. columnare DnaK protein (rDnaK). In year one, three groups of channel catfish (n = 300) were immunized by bath immersion with a live attenuated F. columnare isolate, rDnaK or sham immunized. After 6 weeks, an F. columnare laboratory challenge showed a significant increase in survival (>30%) in both the live attenuated and rDnaK vaccines when compared to the non-immunized control. A rDnaK-specific ELISA revealed significant levels of mucosal IgM antibodies in the skin of catfish immunized with rDnaK at 4- and 6-weeks post immunization. In the second year, three groups of channel catfish (n = 300) were bath immunized with rDnaK alone or with rDnaK after a brief osmotic shock or sham immunized. After 6 weeks a laboratory challenge with F. columnare was conducted and showed a significant increase in survival in the rDnaK (> 25%) and in rDnaK with osmotic shock (>35%) groups when compared to the non-immunized control. The rDnaK-specific ELISA demonstrated significant levels of mucosal IgM antibodies in the skin of catfish groups immunized with rDnaK at 4- and 6-weeks post immunization. To further understand the processes which have conferred immune protection in the rDnaK group, we conducted RNA sequencing of skin samples from the non-immunized (n = 6) and rDnaK treated channel catfish at 1-week (n = 6) and 6 weeks (n = 6) post immunization. Significantly altered gene expression was identified and results will be discussed. Work to further enhance the catfish immune response to F. columnare rDnaK is underway as this protein remains a promising candidate for additional optimization and experimental trials in a production setting.

Evidence that the stress hormone cortisol regulates biofilm formation differently among Flavobacterium columnare isolates.

The impact of cortisol on Flavobacterium columnare biofilm formation was explored. Firstly, the dynamics of biofilm formation by one highly (HV) and one low virulent (LV) F. columnare isolate with and without the stress hormone cortisol under microfluidic flow conditions was characterized. This to confirm that F. columnare cells could form biofilm under cortisol supplementation, and to compare the temporal and structural differences between different treatment groups. One trial revealed that in both isolates cell aggregates resembling biofilms occurred within 7-h post-inoculation. Consequently, cell clusters were sloughed away, followed by a rebuilding of bacterial cell aggregates, suggestive for a high spreading capacity. While the HV isolate revealed cell aggregates formed upstream at all time-points, for the LV isolate this was only seen upon cortisol supplementation. Secondly, the transcriptional effect of genes (gldK, gldL, gldM, gldN, sprA, sprE, sprT, and porV) belonging to the Type IX secretion system involved in gliding motility was investigated in planktonic and biofilm cells of a HV and LV isolate to which no, a low (LD) or high (HD) dose of cortisol was added. Significantly lower expression of gliding genes gldK, gldL, gldM and gldN, and of protein secretion regulator porV was seen in the LV isolate planktonic cells supplemented with a HD-cortisol. The LV isolate biofilm cells treated with the HD-cortisol showed a significant upregulation of sprT, encoding mobile surface adhesion important in bacterial colonization. This is the first evidence for the co-regulatory effect of cortisol on biofilm formation and F. columnare gliding gene expression.

The proliferation and clonal migration of B cells in the systemic and mucosal tissues of channel catfish suggests there is an interconnected mucosal immune system.

IgM transcripts from different mucosal and systemic tissues from a single adult channel catfish have been evaluated. Arrayed heavy chain cDNA libraries from each of these different mucosal and systemic tissues were separately constructed, hybridized with VH family specific probes and a variety of approaches were used to define their structural relationships. Baseline hybridization studies indicated that the tissue libraries had different VH expression patterns, and sequencing studies indicated this was not simply due to varying proportions of the same B cell population. In the systemic tissues of PBL, spleen, and anterior kidney >95% of the sequenced clones in the arrayed libraries represented different heavy chain rearrangements. Diversity was also found in the mucosal libraries of skin, gill lamellae, and two non-adjoining regions of the intestine, but additional populations were identified which indicated localized clonal expansion. Various clonal sets were characterized in detail, and their genealogies indicated somatic mutation accompanied localized clonal expansion with some members undergoing additional mutations and expansion after migration to different mucosal sites. PCR analyses indicated these mucosal clonal sets were more abundant within different mucosal tissues rather than in the systemic tissues. These studies indicate that the mucosal immune system in fish can express B cell transcripts differently from those found systemically. These studies further indicate that the mucosal immune system is interconnected with clonal B cells migrating between different mucosal tissues, results which yield new insight into immune diversity in early vertebrate phylogeny.

Catfish mucus alters the Flavobacterium columnare transcriptome.

Columnaris disease, caused by Flavobacterium columnare, severely impacts the production of freshwater finfish species. Therefore, efforts to better understand the biological processes of F. columnare, including the formation of biofilms and their contribution to disease, are ongoing. In this study, we incubated F. columnare cultures with channel catfish mucus and used high-throughput RNA sequencing to evaluate global changes in gene expression. Our data show that mucus activates in vitro biofilm formation. The analysis of F. columnare transcriptomes after the addition of mucus revealed significant differentially expressed genes (DEGs) between the planktonic and biofilm states. DEGs common among all biofilms were enriched for gene ontology groups including signal transduction, ligand binding and cellular homeostasis and are likely necessary for biofilm formation. Iron acquisition systems included TonB-dependent receptor and ferroxidase genes were expressed among all biofilms, while siderophore synthesis genes were only expressed in mucus-stimulated biofilms. The current analysis of F. columnare transcriptomes adds valuable information about the basic biological processes that occur during the planktonic and biofilm states. This work serves as a basis for future studies on understanding how biofilms are established and how they contribute to disease progression.

The effect of temperature on the mucosal IgM antibody response to DNP-KLH in channel catfish (Ictalurus punctatus).

Bath immersion remains a practical route for immunizing against disease in channel catfish; however research efforts in this area have revealed variable results when activating mucosal Ab responses with different antigens. This is likely due to a number of factors including the individual species, age of the fish, preparation of the immunogens, and differences in the overall dosage and the duration of exposure to vaccines. The current study sought to evaluate the effect of water temperature on the in vivo mucosal adaptive immune response in channel catfish to a protein-hapten antigen, DNP-KLH. Fish were bath immersed at different water temperatures and periodically evaluated over an eighteen week period for the development of serum and mucosal IgM antibodies to DNP-KLH using an indirect enzyme-linked immunosorbent assay. None of the temperature groups produced a serum antibody response; however there were detectable DNP-KLH specific IgM antibodies in the mucus starting at week eight. The extent of the mucosal antibody response and duration differed between the treatments. Our results show that there are intrinsic differences in the capacity to generate in vivo mucosal Ab responses in the skin at different water temperatures and the implications of these findings to channel catfish farming will be discussed.

Repertoire comparison of the B-cell receptor-encoding loci in humans and rhesus macaques by next-generation sequencing.

Rhesus macaques (RMs) are a widely used model system for the study of vaccines, infectious diseases and microbial pathogenesis. Their value as a model lies in their close evolutionary relationship to humans, which, in theory, allows them to serve as a close approximation of the human immune system. However, despite their prominence as a human surrogate model system, many aspects of the RM immune system remain ill characterized. In particular, B cell-mediated immunity in macaques has not been sufficiently characterized, and the B-cell receptor-encoding loci have not been thoroughly annotated. To address these gaps, we analyzed the circulating heavy- and light-chain repertoires in humans and RMs by next-generation sequencing. By comparing V gene segment usage, J-segment usage and CDR3 lengths between the two species, we identified several important similarities and differences. These differences were especially notable in the IgM(+) B-cell repertoire. However, the class-switched, antigen-educated B-cell populations converged on a set of similar characteristics, implying similarities in how each species responds to antigen. Our study provides the first comprehensive overview of the circulating repertoires of the heavy- and light-chain sequences in RMs, and provides insight into how they may perform as a model system for B cell-mediated immunity in humans.

Missing the target: DNAk is a dominant epitope in the humoral immune response of channel catfish (Ictalurus punctatus) to Flavobacterium columnare.

Vaccination remains a viable alternative for bacterial disease protection in fish; however additional work is required to understand the mechanisms of adaptive immunity in the channel catfish. To assess the humoral immune response to Flavobacterium columnare; a group of channel catfish were first immunized with F. columnare LV-359-01 cultured in iron-depleted media, before being challenged with wild type F. columnare LV-359-01. The immunization protocol did not confer increased protection against F. columnare; however both control and immunized responders generated serum and skin IgM antibodies against F. columnare proteins. Western blot analyses of individuals from both groups showed that IgM antibodies were generated to the same 70 kDa extracellular protein, which was identified to be the bacterial chaperonin protein DNAk. Antibodies generated were cross reactive to DNAk proteins found in other gram negative bacteria. Our data suggests that DNAk is the dominant epitope in the channel catfish B-cell response to F. columnare.

Accumulation of VH Replacement Products in IgH Genes Derived from Autoimmune Diseases and Anti-Viral Responses in Human.

VH replacement refers to RAG-mediated secondary recombination of the IgH genes, which renews almost the entire VH gene coding region but retains a short stretch of nucleotides as a VH replacement footprint at the newly generated VH-DH junction. To explore the biological significance of VH replacement to the antibody repertoire, we developed a Java-based VH replacement footprint analyzer program and analyzed the distribution of VH replacement products in 61,851 human IgH gene sequences downloaded from the NCBI database. The initial assignment of the VH, DH, and JH gene segments provided a comprehensive view of the human IgH repertoire. To our interest, the overall frequency of VH replacement products is 12.1%; the frequencies of VH replacement products in IgH genes using different VH germline genes vary significantly. Importantly, the frequencies of VH replacement products are significantly elevated in IgH genes derived from different autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and allergic rhinitis, and in IgH genes encoding various autoantibodies or anti-viral antibodies. The identified VH replacement footprints preferentially encoded charged amino acids to elongate IgH CDR3 regions, which may contribute to their autoreactivities or anti-viral functions. Analyses of the mutation status of the identified VH replacement products suggested that they had been actively involved in immune responses. These results provide a global view of the distribution of VH replacement products in human IgH genes, especially in IgH genes derived from autoimmune diseases and anti-viral immune responses.

VH Replacement Footprint Analyzer-I, a Java-Based Computer Program for Analyses of Immunoglobulin Heavy Chain Genes and Potential VH Replacement Products in Human and Mouse.

VH replacement occurs through RAG-mediated secondary recombination between a rearranged VH gene and an upstream unrearranged VH gene. Due to the location of the cryptic recombination signal sequence (cRSS, TACTGTG) at the 3' end of VH gene coding region, a short stretch of nucleotides from the previous rearranged VH gene can be retained in the newly formed VH-DH junction as a "footprint" of VH replacement. Such footprints can be used as markers to identify Ig heavy chain (IgH) genes potentially generated through VH replacement. To explore the contribution of VH replacement products to the antibody repertoire, we developed a Java-based computer program, VH replacement footprint analyzer-I (VHRFA-I), to analyze published or newly obtained IgH genes from human or mouse. The VHRFA-1 program has multiple functional modules: it first uses service provided by the IMGT/V-QUEST program to assign potential VH, DH, and JH germline genes; then, it searches for VH replacement footprint motifs within the VH-DH junction (N1) regions of IgH gene sequences to identify potential VH replacement products; it can also analyze the frequencies of VH replacement products in correlation with publications, keywords, or VH, DH, and JH gene usages, and mutation status; it can further analyze the amino acid usages encoded by the identified VH replacement footprints. In summary, this program provides a useful computation tool for exploring the biological significance of VH replacement products in human and mouse.