Characterization of Hagfish (Eptatretus burgeri) Variable Lymphocyte Receptor-Based Antibody and Its Potential Role in the Neutralization of Nervous Necrosis Virus.

The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey (Petromyzon marinus) and hagfish (Eptatretus burgeri). Hagfish VLRBs are composed of leucine-rich repeat (LRR) modules, conjugated with a superhydrophobic C-terminal tail, which contributes to low levels of expression in recombinant protein technology. In this study, we screened Ag-specific VLRBs from hagfish immunized with nervous necrosis virus (NNV). The artificially multimerized form of VLRB was constructed using a mammalian expression system. To enhance the level of expression of the Ag-specific VLRB, mutagenesis of the VLRB was achieved in vitro through domain swapping of the LRR C-terminal cap and variable LRR module. The mutant VLRB obtained, with high expression and secretion levels, was able to specifically recognize purified and progeny NNV, and the Ag binding ability of this mutant was increased by at least 250-fold to that of the nonmutant VLRB. Furthermore, preincubation of the Ag-specific VLRB with NNV reduced the infectivity of NNV in E11 cells in vitro, and in vivo experiment. Our results suggest that the newly developed Ag-specific VLRB has the potential to be used as diagnostic and therapeutic reagents for NNV infections in fish.

Membrane vesicles from antibiotic-resistant Staphylococcus aureus transfer antibiotic-resistance to antibiotic-susceptible Escherichia coli.

AIM: Bacteria naturally produce membrane vesicles (MVs), which have been shown to contribute to the spread of multi-drug resistant bacteria (MDR) by delivering antibiotic-resistant substances to antibiotic-susceptible bacteria. Here, we aim to show that MVs from Gram-positive bacteria are capable of transferring ß-lactam antibiotic-resistant substances to antibiotic-sensitive Gram-negative bacteria. MATERIALS AND METHODS: MVs were collected from a methicillin-resistant strain of Staphylococcus aureus (MRSA) and vesicle-mediated fusion with antimicrobial-sensitive Escherichia coli (RC85). It was performed by exposing the bacteria to the MVs to develop antimicrobial-resistant E. coli (RC85-T). RESULTS: The RC85-T exhibited a higher resistance to ß-lactam antibiotics compared to the parent strain. Although the secretion rates of the MVs from RC85-T and the parent strain were nearly equal, the ß-lactamase activity of the MVs from RC85-T was 12-times higher than that of MVs from the parent strain, based on equivalent protein concentrations. Moreover, MVs secreted by RC85-T were able to protect ß-lactam-susceptible E. coli from ß-lactam antibiotic-induced growth inhibition in a dose-dependent manner. CONCLUSION: MVs play a role in transferring substances from Gram-positive to Gram-negative bacteria, shown by the release of MVs from RC85-T that were able to protect ß-lactam-susceptible bacteria from ß-lactam antibiotics. SIGNIFICANCE AND IMPACT OF STUDY: MVs are involved in the emergence of antibiotic-resistant strains in a mixed bacterial culture, helping us to understand how the spread of multidrug-resistant bacteria could be reduced.

Mediating the effects of depression in the relationship between university students' attitude toward suicide, frustrated interpersonal needs, and non-suicidal self-injury during the COVID-19 pandemic.

OBJECTIVE: The study aimed to examine the relationship on attitudes toward suicide, frustrated interpersonal needs, and non-suicidal self-injury (NSSI) of the university students. METHODS: The participants included 175 university students. Data were analyzed using the SPSS PROCESS macro (Model 4). RESULTS: Depression showed a fully mediating effect on the relationship between one's attitude toward suicide and NSSI behaviors. Furthermore, depression showed a full mediating impact on the relationship between frustrated interpersonal needs and NSSI behaviors. CONCLUSIONS: These findings indicate that suicidal attitudes and frustrated interpersonal needs should be considered significant factors for developing NSSI preventions and intervention among university students.

Potential Use of Genetically Engineered Variable Lymphocyte Receptor B Specific to Avian Influenza Virus H9N2.

The variable lymphocyte receptor (VLR) B of jawless vertebrates functions as a secreted Ab of jawed vertebrates and has emerged as an alternative Ab with a single polypeptide chain. After observing an upregulated VLRB response in hagfish immunized with avian influenza virus (AIV) subtype H9N2, we screened AIV H9N2-specific VLRB using a mammalian expression system. To improve the binding avidity of the Ag-specific VLRB to the Ag, we enabled multimerization of the VLRB by conjugating it with C-terminal domain of human C4b-binding protein. To dramatically enhance the expression and secretion of the Ag-specific VLRB, we introduced a glycine-serine linker and the murine Ig κ leader sequence. The practical use of the Ag-specific VLRB was also demonstrated through various immunoassays, detected by anti-VLRB Ab (11G5). Finally, we found that the Ag-specific VLRB decreased the infectivity of AIV H9N2. Together, our findings suggest that the generated Ag-specific VLRB could be used for various immunoapplications.

The Importance of Porins and ß-Lactamase in Outer Membrane Vesicles on the Hydrolysis of ß-Lactam Antibiotics.

Gram-negative bacteria have an outer membrane inhibiting the entry of antibiotics. Porins, found within the outer membrane, are involved in regulating the permeability of ß-lactam antibiotics. ß-lactamases are enzymes that are able to inactivate the antibacterial properties of ß-lactam antibiotics. Interestingly, porins and ß-lactamase are found in outer membrane vesicles (OMVs) of ß-lactam-resistant Escherichia coli and may be involved in the survival of susceptible strains of E. coli in the presence of antibiotics, through the hydrolysis of the ß-lactam antibiotic. In this study, OMVs isolated from ß-lactam-resistant E. coli and from mutants, lacking porin or ß-lactamase, were evaluated to establish if the porins or ß-lactamase in OMVs were involved in the degradation of ß-lactam antibiotics. OMVs isolated from E. coli deficient in ß-lactamase did not show any degradation ability against ß-lactam antibiotics, while OMVs lacking OmpC or OmpF showed significantly lower levels of hydrolyzing activity than OMVs from parent E. coli. These data reveal an important role of OMVs in bacterial defense mechanisms demonstrating that the OmpC and OmpF proteins allow permeation of ß-lactam antibiotics into the lumen of OMVs, and antibiotics that enter the OMVs can be degraded by ß-lactamase.

Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus.

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.

High Efficiency Flip Chip-Based UV Emitter with Indium Tin Oxide Nano Grains/Al Reflector and Periodic Microhole Arrays.

We propose a high efficiency flip chip-based ultraviolet (UV) emitter with aluminum (Al) reflector that includes indium tin oxide (ITO) nano grains for current injection between the Al and p-AlGaN layer. Al has attracted attention as a reflector for high efficiency UV emitters because of its high reflectance in the UV region. To improve the efficiency of UV emitter, we generated periodic microhole arrays on the p-AlGaN layer, which serve as a scattering center in the flip chip structure and enhance the light extraction efficiency. The light output power of the fabricated flip chip-based UV emitter with ITO nano grains/Al reflector and microhole arrays on the p-AlGaN layer is significantly improved by 72% and 45% at an injection current of 20 mA, compared to that of UV emitter with only Al reflector and ITO nano grains/Al reflector.

Transparent Conductive Electrodes of ß-Ga2O3/Ag/ß-Ga2O3 Multilayer for Ultraviolet Emitters.

We investigated the optical and electrical properties of a ß-Ga2O3/Ag/ß-Ga2O3 multilayer transparent conductive electrode deposited on an α-Al2O3 (0001) substrate. For the deposition of a continuous Ag layer, we preliminarily performed anultraviolet-ozone pretreatment of the Ga2O3 bottom layer. To obtain a stable ß-phase of Ga2O3, the ß-Ga2O3/Ag/ß-Ga2O3 multilayer was annealed at 700 °C under N2 atmosphere. The transmittance and sheet resistance of the ß-Ga2O3/Ag/ß-Ga2O3 multilayer were critically affected by the surface morphology and thickness of the Ag interlayer. The multilayer with optimized thicknesses (ß-Ga2O3 top layer: 30 nm; Ag interlayer: 12 nm; ß-Ga2O3 bottom layer: 60 nm) exhibited a resistance of 8.48 Ωsq-1, an average optical transmittance of 87.16% in the ultraviolet wavelength range from 300 to 350 nm, and a figure of merit of 29.81 × 10-3 Ω-1.

Effects of Angelica gigas Nakai on the production of decursin- and decursinol angelate-enriched eggs.

BACKGROUND: The livestock industry requires high-quality products, as well as improved productivity. There have been many studies regarding the utilization of feed additives aiming to increase productivity, enhance immune functions and prevent infectious diseases in livestock. Biofunctional feed additives would be beneficial not only for animal health, but also for consumers. In the present study, we utilized root and byproduct (stem and leaf) powders of Angelica gigas Nakai (AGN, Korean Danggui) as feed additives and examined the deposition of biofunctional compounds, such as decursin and decursinol angelate, into egg white and yolk. RESULTS: We optimized the detection system for decursin and decursinol angelate, and determined the amounts of decursin and decursinol angelate derived from AGN byproducts (stem and leaf) as well as root. In Experiment 1, laying hens were fed with the dried AGN root powder and the effective compounds were detected in egg white and yolk. Subsequently, in Experiment 2, we examined AGN byproducts as an alternative feeding supplement. Additionally, biochemical parameters were analyzed to evaluate changes in the health of the hens by feeding AGN root powder. The results obtained indicated that decursin and decursinol angelate were stably transferred into egg white and yolk by feeding AGN byproducts as well as root. Intriguingly, plasma cholesterol levels were significantly decreased in a dose-dependent manner, and those of interleukin-1ß, as an immune-related biomarker, were considerably increased in the treated hens. CONCLUSION: These results indicated that AGN root and byproducts (stem and leaf) could be utilized for the production of value-added eggs and improving the health of hens in the poultry industry. © 2018 Society of Chemical Industry.

Wide Bandgap Transparent Conducting Electrode of FTO/Ag/FTO Structure for Ultraviolet Light-Emitting Diodes.

We investigated the effect of the Ag interlayer thickness on the structural, electrical and optical properties of FTO/Ag/FTO structures designed for use in wide bandgap transparent conducting electrodes. The top and bottom FTO layers were deposited on α-Al2O3 (0001) substrates via RF magnetron sputtering at 300 °C and Ag interlayers were deposited using an e-beam evaporator system. We optimized the figure of merit by changing the thickness of the inserted Ag interlayer from 10 nm to 14 nm, achieving a maximum value of 2.46 × 10-3 Ω-1 and a resistivity of 6.4 × 10-4 Ω · cm using an FTO (70 nm)/Ag (14 nm)/FTO (40 nm) structure. Furthermore, the average optical transmittance in the deep UV range (300 to 330 nm) was 82.8%.

Enhancement of glycoprotein-based DNA vaccine for viral hemorrhagic septicemia virus (VHSV) via addition of the molecular adjuvant, DDX41.

The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been thoroughly studied in recent years. Glycoprotein (G)-based DNA vaccines had been proven to be effective in combating infection against Rhabdovirus (especially infectious hematopoietic necrosis virus, IHNV) in salmonids. DDX41 is a helicase known to induce antiviral and inflammatory responses by inducing a type I IFN innate immune response. To gain more information regarding G-based DNA vaccines in olive flounder (Paralicthys olivaceus), we tried to develop a more efficient G-based DNA vaccine by adding a molecular adjuvant, DDX41. We designed a DNA vaccine in which the VHSV glycoprotein (G-protein) and DDX41 were driven by the EF-1α and CMV promoters, respectively. Olive flounders were intramuscularly immunized with 1 µg of plasmids encoding the G-based DNA vaccine alone (pEF-G), the molecular adjuvant alone (pEF-D), or the vaccine-adjuvant construct (pEF-GD). At two different time points, 15 and 30 days later, the fish were intraperitoneally infected with VHSV (100 µL; 1 × 106 TCID50/mL). Our assays revealed that the plasmid constructs showed up-regulated expression of IFN-1 and its associated genes at day 3 post-vaccination in both kidney and spleen samples. Specifically, pEF-GD showed statistically higher expression of immune response genes than pEF-G and pEF-D treated group (p < 0.05/p < 0.001). After VHSV challenge, the fish group treated with pEF-GD showed higher survival rate than the pEF-G treated group, though difference was not statistically significant in the 15 dpv challenged group however in the 30 dpv challenged group, the difference was statistically significant (p < 0.05). Together, these results clearly demonstrate that DDX41 is an effective adjuvant for the G-based DNA vaccine in olive flounder. Our novel findings could facilitate the development of more effective DNA vaccines for the aquaculture industry.

Development of a monoclonal antibody against the CD3ε of olive flounder (Paralichthys olivaceus) and its application in evaluating immune response related to CD3ε.

The T cell receptor (TCR) is the binding site of antigen and is responsible for specifically activating the adaptive immune response. CD3, an essential component of the CD3-TCR complex, is known to be composed of γδ and ε chains in teleost. However, there are few monoclonal antibodies (mAb) available to identify these molecules on T cells, so we aimed to produce a mAb against CD3ε to improve our understanding of T cell immune response in olive flounder (Paralichthys olivaceus). CD3ε recombinant protein was expressed in yeast, the expression of which was confirmed by SDS-PAGE, MALDI-TOF/TOF MS and Western blot analysis. A CD3ε-specific mAb 4B2 was selected, the specificity of which was examined by confocal microscopy, flow cytometry and RT-PCR, and the mAb was subsequently used to examine the CD3ε lymphocyte population in several different immune organs, with relatively high percentages of these cells seen in trunk-kidney and spleen, while lower percentages were seen in the liver and peripheral blood of olive flounder. During a viral hemorrhagic septicemia virus (VHSV) infection in olive flounder, the number of CD3ε lymphocytes was seen to gradually increase in the liver, spleen and trunk-kidney of infected fish until 7 days post infection (dpi). In peripheral blood, on the other hand, the increase in CD3ε lymphocyte numbers peaked by 3 dpi. These results suggest that CD3ε lymphocytes might be involved in the immune response against VHSV.

C-X-C chemokine receptor type 4 (CXCR4) is a key receptor for chicken primordial germ cell migration.

In mammals, germ cells originate outside of the developing gonads and follow a unique migration pattern through the embryonic tissue toward the genital ridges. Many studies have attempted to identify critical receptors and factors involved in germ cell migration. However, relatively few reports exist on germ cell receptors and chemokines that are involved in germ cell migration in avian species. In the present study, we investigated the specific migratory function of C-X-C chemokine receptor type 4 (CXCR4) in chicken primordial germ cells (PGCs). We induced loss-of-function via a frameshift mutation in the CXCR4 gene in chicken PGCs using clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. The migratory capacity of CXCR4 knockout PGCs was significantly reduced in vivo after transplantation into recipient embryos. However, CXCR4-expressing somatic cell lines, such as chicken DT40 and DF1, failed to migrate into the developing gonads, suggesting that another key factor(s) is necessary for targeting and settlement of PGCs into the genital ridges. In conclusion, we show that CXCR4 plays a critical role in the migration of chicken germ cells.

Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells.

OBJECTIVE: Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. METHODS: Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2) gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7) cells during muscle differentiation. RESULTS: Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO) QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. CONCLUSION: We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

Efficient transgene expression system using a cumate-inducible promoter and Cre-loxP recombination in avian cells.

OBJECTIVE: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. METHODS: After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. RESULTS: In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. CONCLUSION: Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression.

Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect.

OBJECTIVE: Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells. METHODS: Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. RESULTS: Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. CONCLUSION: The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

Myotube differentiation in clustered regularly interspaced short palindromic repeat/Cas9-mediated MyoD knockout quail myoblast cells.

OBJECTIVE: In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation can be applied to improve traits such as growth and meat production. We investigated the regulatory pathway of MyoD and its role in muscle differentiation in quail myoblast cells. METHODS: The MyoD gene was mutated by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology and single cell-derived MyoD mutant sublines were identified to investigate the global regulatory mechanism responsible for muscle differentiation. RESULTS: The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (MyoD KO QM7#4) through single cell pick-up and expansion. In the undifferentiated condition, paired box 7 expression in MyoD KO QM7#4 cells was not significantly different from regular QM7 (rQM7) cells. During differentiation, however, myotube formation was dramatically repressed in MyoD KO QM7#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD KO QM7#4 cells even after an extended differentiation period. These results indicate that MyoD is critical for muscle differentiation. Furthermore, we analyzed the global regulatory interactions by RNA sequencing during muscle differentiation. CONCLUSION: With CRISPR/Cas9-mediated genomic editing, single cell-derived sublines with a specific knockout gene can be adapted to various aspects of basic research as well as in functional genomics studies.

Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene.

OBJECTIVE: Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise. METHODS: We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR). RESULTS: Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms. CONCLUSION: It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.

Serotype-identifying ions in Listeria monocytogenes using matrix-associated laser desorption ionization-time of flight mass spectrometry.

Listeria monocytogenes is a foodborne pathogen that can cause a potentially life-threatening infection, and almost all cases of human listeriosis are caused by L. monocytogenes isolates in serotypes 1/2a, 1/2b, 1/2c, and 4b. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In the current study, we examined the potential of MALDI-TOF MS for rapid identification of the foodborne pathogen L. monocytogenes and to identify high-risk serotypes. To achieve this, MALDI-TOF MS was applied to 50 L monocytogenes strains. All strains were identified as L. monocytogenes species based on pattern matching against reference spectra for the species. Importantly, 83 specific mass ions were consistently and uniquely found in high-risk L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b. These 83 mass ions were also unique to specific combinations of these serotypes, which enabled specific identification of these four serotypes using MALDI Biotyper analysis. Hence, this method shows potential for using MALDI-TOF MS for the rapid identification of L. monocytogenes species and to discriminate high-risk L. monocytogenes serotypes through specific serotype-specific biomarker ions.

Passive Immunization with Recombinant Antibody VLRB-PirAvp/PirBvp-Enriched Feeds against Vibrio parahaemolyticus Infection in Litopenaeus vannamei Shrimp.

The causative agent of acute hepatopancreatic necrosis disease (AHPND) is the bacterium, Vibrio parahaemolyticus, which secretes toxins into the gastrointestinal tract of its host. Vibrio parahaemolyticus toxins A and B (PirAvp/PirBvp) have been implicated in the pathogenesis of this disease, and are, therefore, the focus of studies developing treatments for AHPND. We previously produced recombinant antibodies based on the hagfish variable lymphocyte receptor B (VLRB) capable of neutralizing some viruses, suggesting that this type of antibody may have a potential application for treatment of AHPND. Here, recombinant PirAvp/PirBvp, produced using a bacterial expression system, were used as antigens to screen a hagfish VLRB cDNA library to obtain PirAvp/PirBvp-specific antibodies. A cell line secreting these antibodies was established by screening and cloning the DNA extracted from hagfish B cells. Supernatants collected from cells secreting the PirAvp/PirBvp antibodies were collected and concentrated, and used to passively immunize shrimp to neutralize the toxins PirAvp or PirBvp associated with AHPND. Briefly, 10 µg of PirAvp and PirBvp antibodies, 7C12 and 9G10, respectively, were mixed with the shrimp feed, and fed to shrimp for three days consecutive days prior to experimentally infecting the shrimp with V. parahaemolyticus (containing toxins A and B), and resulting mortalities recorded for six days. Results showed significantly higher level of survival in shrimp fed with the PirBvp-9G10 antibody (60%) compared to the group fed the PirAvp-7C12 antibody (3%) and the control group (0%). This suggests that VLRB antibodies may be a suitable alternative to immunoglobulin-based antibodies, as passive immunization treatments for effective management of AHPND outbreaks within shrimp farms.