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• F. columnare recombinant DnaK protein vaccine with or without an immersion adjuvant confers protection against columnaris disease. • The benefit of using an immersion adjuvant is the reduction of immunization time required to achieve that protection. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • IgM transcripts from catfish show different B cell populations in tissues. • Clonal B cells reveal that somatic mutation accompanied localized clonal expansion. • Mucosal B cell transcripts can be expressed differently from that found systemically. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

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. • RNA sequencing performed to evaluate the shrimp host response to Vibrio toxin. • Differential gene expression identified in the shrimp hepatopancreas post-exposure to Vibrio toxin. • The shrimp host response activated regulatory pathways, including cell survival signaling and innate immune processes. [ABSTRACT FROM AUTHOR]

Abstract: VH replacement occurs through RAG-mediated secondary recombination to change unwanted IgH genes and diversify antibody repertoire. The biological significance of VH replacement remains to be explored. Here, we show that VH replacement products are highly enriched in IgH genes encoding anti-HIV antibodies, including anti-gp41, anti-V3 loop, anti-gp120, CD4i, and PGT antibodies. In particular, 73% of the CD4i antibodies and 100% of the PGT antibodies are encoded by potential VH replacement products. Such frequencies are significantly higher than those in IgH genes derived from HIV infected individuals or autoimmune patients. The identified VH replacement products encoding anti-HIV antibodies are highly mutated; the VH replacement “footprints” within CD4i antibodies preferentially encode negatively charged amino acids within the IgH CDR3; many IgH encoding PGT antibodies are likely generated from multiple rounds of VH replacement. Taken together, these findings uncovered a potentially significant contribution of VH replacement products to the generation of anti-HIV antibodies. [Copyright &y& Elsevier]

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. • Recombinant proteins rPirA and rPirB were produced to evaluate their relative toxicities in shrimp. • Both rPirA and rPirB were potent in causing acute death but combination of the two was more deadly. • Formation of an octamer rPirA was observed, suggesting intra- and inter-molecule interaction. [ABSTRACT FROM AUTHOR]