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.

Elucidating the Functional Roles of Helper and Cytotoxic T Cells in the Cell-Mediated Immune Responses of Olive Flounder (Paralichthys olivaceus).

In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder (Paralichthys olivaceus) CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder's CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.

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.

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.

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.

The cytosolic sensor, DDX41, activates antiviral and inflammatory immunity in response to stimulation with double-stranded DNA adherent cells of the olive flounder, Paralichthys olivaceus.

DDX41, a receptor belonging to the DExD family, functions as a DNA sensor in the mammalian cytoplasm and mediates the antiviral response in host cells. Here, the olive flounder DDX41 was found to have 2267-bp long and encodes a putative protein of 614 amino acid residues. The olive flounder DDX41 mRNA was presented in all tested tissues, and was distinctly expressed in fish naturally infected with LCDV. High expression levels were observed in the heart, liver, kidney and stomach. Furthermore, the olive flounder DDX41 mRNA expression increased significantly in adherent (monocyte-like) cells following stimulation with a DNA virus. Reporter assays showed that the transcriptional activity of the IFN-I promoter was enhanced in DDX41-overexpressing HINAE cells treated with C-di-GMP (dinucleotides). Overexpression of DDX41 also induced the antiviral and inflammatory cytokine gene expression through cytoplasmic C-di-GMP treatment. These results suggest that DDX41 functions as a cytosolic DNA sensor that is capable of inducing antiviral activity and inflammatory responses in the olive flounder.

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.

Poly (I:C)-Potentiated Vaccination Enhances T Cell Response in Olive Flounder (Paralichthys olivaceus) Providing Protection against Viral Hemorrhagic Septicemia Virus (VHSV).

Viral hemorrhagic septicemia (VHS), caused by viral hemorrhagic septicemia virus (VHSV), is a viral disease affecting teleosts, and is the major cause of virus-related deaths in olive flounder (Paralichthys olivaceus). Research has focused on ways to control VHS, and recently, the use of polyinosinic-polycytidylic acid poly (I:C)-potentiated vaccination has been investigated, whereby fish are injected with poly (I:C) and then with live pathogenic virus, resulting in a significant decrease in VHSV-related mortality. T cell responses were investigated in the present study after vaccinating olive flounder with poly (I:C)-potentiated vaccination to understand the ability of poly (I:C) to induce T cell immunity. Stimulation of T cell responses with the poly (I:C)-potentiated vaccination was confirmed by examining levels of CD3+ T cells, CD4-1+ T cells and CD4-2+ T cells. Higher levels of CD4-2+ T cells were found in vaccinated fish than CD4-1+ T cells, believed to result from a synergistic effect between poly (I:C) administration and pathogenic VHSV immunization. More importantly, the role of CD4-2+ T cells in the antiviral response was clearly evident. The results of this study suggest that the outstanding protection obtained with the poly (I:C)-potentiated vaccination is due to the robust immune response initiated by the CD4-2+ T cells.

Involvement of CD4-1 T cells in the cellular immune response of olive flounder (Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and nervous necrosis virus (NNV) infection.

The occurrence of CD4 helper T cells has already been established for a number of teleost species, though, it has not been possible to analyze these responses at a cellular level due to a large lack of appropriate monoclonal antibodies (mAbs). In the present study, we produced a mAb against olive flounder (Paralichthys olivaceus) CD4-1 lymphocyte to investigate the functional activity of the cells to improve our understanding of the T cell response in this species. This mAb is specifically able to detect CD4-1 lymphocytes in olive flounder proved by immunofluorescence staining and RT-PCR analysis. In flow cytometry analysis, the number of CD4-1-positive lymphocytes was observed to gradually increase from 3 days post infection (dpi) and then reach peak at 7 dpi against two viruses challenge. As a conclusion, both the basic properties of CD4-1 T cells and its response to viral infections in olive flounder are very similar to the helper T cells in terrestrial animals.

Antibiotic susceptibility and resistance of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus).

The rates of antibiotic susceptibility and resistance were investigated in Streptococcus iniae and Streptococcus parauberis isolates obtained from diseased olive flounders (Paralichthys olivaceus) collected from fish farms in Jeju Island, Korea. Isolates of S. iniae (n=65) were susceptible to cefotaxime, erythromycin, ofloxacin, penicillin, tetracycline and vancomycin, as demonstrated by the minimum inhibitory concentration (MIC) test. Isolates of S. parauberis (n=86) were highly resistant to erythromycin (58% of the 86 isolates tested) and tetracycline (63% of the 86 isolates tested). Fifty-four isolates of tetracycline-resistant S. parauberis contained the tet(M/O/S) genes, of which 39 and 12 isolates contained the tet(M) and tet(S) genes, respectively, whereas 3 isolates contained both the tet(M) and tet(S) genes. Among the erythromycin-resistant isolates of S. parauberis (n=50) only 14 contained the erm(B) gene. These results suggest that the tet(S) and erm(B) genes of S. parauberis are involved in the acquisition of high-level resistance to erythromycin and tetracycline. Our findings reveal a high rate of antibiotic resistance among strains of S. parauberis and emphasize the need to develop an appropriate vaccine to reduce the use of antibiotics.

Development of a modified yeast display system for screening antigen-specific variable lymphocyte receptor B in hagfish (Eptatretus burgeri).

The variable lymphocyte receptor B (VLRB) of jawless vertebrates has a similar function to the antibodies produced by jawed vertebrates, and has been considered as an alternative source to mammalian antibodies for use in biological research. We developed a modified yeast display vector system (pYD8) to display recombinant hagfish VLRB proteins on the extracellular surface of yeast for the isolation of antigen-specific VLRBs. After observing an up-regulation in the VLRB response in hagfish immunized with hemagglutinin 1 of avian influenza virus H9N2 subtype (H9N2-HA1), the antigen-specific VLRBs decorated on the yeast's surface were selected by quantitative library screening through magnetic-activated cell sorting (MACS) and fluorescent-activated cell sorting (FACS). We also demonstrated a strong specificity of the antigen-specific VLRBs, when expressed as a secreted protein using a mammalian expression system. Together, our findings suggest that the pYD8 vector system could be useful for screening antigen-specific hagfish VLRBs, and the specificity of secreted VLRB may have potential for a variety of biological applications.

Generation and characterization of hagfish variable lymphocyte receptor B against glycoprotein of viral hemorrhagic septicemia virus (VHSV).

Variable lymphocyte receptors B (VLRBs) are non-immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C-terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b-binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen-specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen-binding properties by at least 125-fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.

Globular-shaped variable lymphocyte receptors B antibody multimerized by a hydrophobic clustering in hagfish.

In hagfish and lampreys, two representative jawless vertebrates, the humoral immunity is directly mediated by variable lymphocyte receptors B (VLRBs). Both monomeric VLRBs are structurally and functionally similar, but their C-terminal tails differ: lamprey VLRB has a Cys-rich tail that forms disulfide-linked pentamers of dimers, contributing to its multivalency, whereas hagfish VLRB has a superhydrophobic tail of unknown structure. Here, we reveal that VLRBs obtained from hagfish plasma have a globular-shaped multimerized form (approximately 0.6 to 1.7 MDa) that is generated by hydrophobic clustering instead of covalent linkage. Electron microscopy (EM) and single-particle analysis showed that the multimerized VLRBs form globular-shaped clusters with an average diameter of 28.7 ± 2.2 nm. The presence of VLRBs in the complex was confirmed by immune-EM analysis using an anti-VLRB antibody. Furthermore, the hydrophobic hagfish C-terminus (HC) was capable of triggering multimerization and directing the cellular surface localization via a glycophosphatidylinositol linkage. Our results strongly suggest that the hagfish VLRB forms a previously unknown globular-shaped antibody. This novel identification of a structurally unusual VLRB complex may suggest that the adaptive immune system of hagfish differs from that of lamprey.

Application of immunoproteomics in developing a Streptococcus iniae vaccine for olive flounder (Paralichthys olivaceus).

Streptococcus iniae is the major etiological agent of streptococcosis, which is responsible for hemorrhagic septicemia in fish, particularly olive flounder (Paralichthys olivaceus). In the present study, we sought to understand the pathogenicity and immunogenicity of S. iniae in order to develop a vaccine for streptococcosis. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting, was employed to investigate the pathogenicity and immunogenicity of two S. iniae isolates, Jeju-13 and Jeju-45, in olive flounder. The virulence of Jeju-13 was moderate whereas that of Jeju-45 was high. A vaccination trial with formalin-killed Jeju-45 demonstrated relatively low protection against the homologous isolate compared with the heterologous isolate. A significant difference in the secretion of extracellular products (ECPs) was noticed between the two S. iniae isolates. ECP antigens were highly immunogenic compared to those from whole cell lysates as determined by 2-DE immunoblot assay of Jeju-13 and Jeju-45 anti-sera collected from post-challenge survival fish. Furthermore, there were differences in the appearance of antigenic spots on 2-DE immunoblot profiles of ECPs of the respective sera. Interestingly, the mixture of killed-cells and concentrated ECPs from Jeju-45 led to significant protection against the homologous isolate of S. iniae in olive flounder. The present study demonstrates the usefulness of immunoproteomics in understanding the pathogenicity of S. iniae to aid the development of a vaccine for fish streptococcosis.

Partial two-dimensional gel electrophoresis (2-DE) maps of Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408.

To examine the proteomes of 2 important causative agents of fish streptococcosis, Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408, we used 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry to generate 2-DE maps of these type strains. Silver-stained 2-DE gels of S. iniae ATCC29178 and L. garvieae KG9408 revealed approximately 320 and 300 spots, respectively, and immobilized pH gradient strips (13 cm, pH 4 to 7) revealed that the majority of the detected spots were concentrated in the pH range of 4.5 to 5.5. The spots were randomly selected from the 2-DE profiles and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. The majority of the identified proteins were functionally related to energy and carbohydrate metabolism (e.g. enolase ATPase, glyceraldehyde-3-phosphate dehydrogenase) or translation and translocation (e.g. elongation factor G, elongation factor Tu, DNA-directed RNA polymerase alpha chain). These data, along with our partial 2-DE maps of S. iniae ATCC29178 and L. garvieae KG9408, may help suggest antigenic proteins for the development of effective diagnostic tools and vaccines against S. iniae and L. garvieae.

Investigation of variable lymphocyte receptors in the alternative adaptive immune response of hagfish.

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites.

Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M(r) between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin alpha- and beta-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.

Comparison of proteome and antigenic proteome between two Neospora caninum isolates.

This study was conducted to explore the relationship between two isolates of Neospora caninum (N. caninum) (KBA-2 and VMDL-1) using proteomics. To achieve the goal, proteins of N. caninum tachyzoite lysates of KBA-2 and VMDL-1 were separated by two-dimensional gel electrophoresis (2-DE), stained with silver-nitrate and analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to compare protein profiles. In addition, proteins separated by 2-DE were transferred to membranes, probed with bovine anti-N. caninum KBA-2 immunoglobulin G, and reactive proteins were visualized and compared between the two isolates. Most spots on 2-DE profiles and antigenic spots on 2-DE immunoblot profiles were located at similar locations in terms of isoelectric point and molecular weight. Proteins common to both isolates included the following: heat shock protein 70, subtilisin-like serine protease, nucleoside triphosphatase, heat shock protein 60, pyruvate kinase, tubulin alpha, tubulin beta, enolase, putative protein disulfide isomerase, actin, fructase-1,6-bisphosphatase, putative ribosomal protein S2, microneme protein Nc-P38, lactate dihydrogenase, fructose-1,6-bisphosphatase aldolase, serine threonine phosphatase 2C, 14-3-3 protein homologue, N. caninum dense granule-1 and NcGRA2. As a consequence, even though N. caninum KBA-2 and VMDL-1 isolates were isolated from geographically distinct locations there were significant homology in the proteome and antigenic proteome profiles. In addition, proteomic approach was verified as a useful tool for understanding of host immune response against different isolates of protozoa.