Comparison of Anti-rabies Virus Nucleoprotein IgY Prepared by DNA Immunization and Protein Immunization.

Immunization of egg-laying hens with viral antigens efficiently produces large amounts of virus-specific IgY antibodies from egg yolks. A supply of practical and economical antibodies against the rabies virus is being desired worldwide. We immunized hens with the antigen gene DNA of the rabies virus, purified specific IgY antibodies from the egg yolk, and characterized the immuno-protein chemistry for use as a diagnosis. To prepare specific IgY antibodies against rabies virus nucleoprotein (RV-N) by DNA immunization, laying hens were pre-injected with λ-carrageenan or Freund's complete adjuvant to increase local immune activity (pre-immune stimulation), and then immunized with RV-N recombinant plasmid DNA. RV-N-specific IgY antibodies were prepared from egg yolks of immunized hens. For comparison, conventional protein antigen immunization was also used to induce the production of RV-N-specific IgY antibodies. Laying hens were immunized with an RV-N protein antigen and RV-N-specific IgY was purified from egg yolks. The binding activity against RV-N antigens was examined using IgY samples prepared by DNA (with pre-immune stimulation) and protein immunization. Immunohistochemical staining showed that IgY antibodies prepared by protein immunization strongly detected viral antigens in the brain sections of dogs infected with the virus, whereas IgY antibodies prepared by DNA immunization did not. Enzyme-linked immunosorbent assay was performed using a commercially available rabies vaccine (inactivated virus) treated with 10% formalin and heating (60°C, 30 min and 90°C, 5 min). IgY prepared by DNA immunization had weaker reactivity with denatured antigens and lower antigen concentrations than IgY prepared by protein immunization. These results suggest that it is necessary to develop a DNA immunization method for inducing IgY antibodies against the rabies virus that strongly bind to native and denatured antigens to prepare specific IgYs that can be used for antigen detection in clinical tests.

Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants.

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund's complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16-19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.

A Comparative Study on Egg Yolk IgY Production with Different Adjuvants and their Inhibitory Effects on Staphylococcus aureus.

Objectives: Atopic dermatitis (AD) is one of the most common skin disorders in infants and children and is often aggravated by increased Staphylococcus aureus (S. aureus) colonization. An inhibitory effect of a specific egg yolk antibody (IgY) on S. aureus growth was demonstrated in this study. Furthermore, the effects of water- or oil-based adjuvants on the preparation of anti-S. aureus IgY and hen immunization were compared. Methods: Hens were immunized intramuscularly with formalin-killed S. aureus mixed with either a water-soluble polysaccharide λ-carrageenan, oil-based Freund's complete adjuvant (FCA), or Freund's incomplete adjuvant (FIA). Anti-S. aureus IgYs (FIA-IgY, FCA/FIA-IgY, and λCarra-IgY) were purified from the egg yolk of immunized hen eggs, and the activity of the IgY against S. aureus antigen was measured by ELISA. The proportion of each IgY that was absorbed by S. aureus was also determined. Then, the effect of purified anti-S. aureus IgY on S. aureus growth inhibition was investigated in vitro. Results: The yolk of eggs and purified FIA-IgY from the FIA group showed the highest antibody activity, followed by FCA/FIA-IgY and λCarra-IgY. The proportion of each IgY that was absorbed by S. aureus antigen was as follows: FIA-IgY (18.1%), FCA/FIA-IgY (12.9%), and λCarra-IgY (7.0%). Only FIA-IgY significantly inhibited S. aureus growth in liquid medium. Conclusion: A specific IgY that was produced using the FIA adjutant inhibited S. aureus growth. Although water-soluble λ-carrageenan showed an adjuvant effect on anti-S. aureus IgY induction in egg yolk, but did not inhibit S. aureus growth. The use of the oil adjuvant FIA was necessary in the preparation of anti-S. aureus IgY as a treatment for AD symptoms.

Spatial Analysis of Phosphatidylinositol Molecular Species in Pork Chop Tissues Using Matrix-assisted Laser Desorption/ionization-Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualizing lipids in biological tissues. Phosphatidylinositol (PI), a phospholipid in pork, is a major source of inositol in animal-derived foods believed to be protective against diseases related to pregnancy and cancer. However, the distribution of PI molecular species in pork is not well understood. Here, we performed MALDI-MSI analysis to investigate the distribution and composition of PI molecular species in pork chop comprising Longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle. Twelve diacyl-PI molecular species were identified using liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) and MALDI-MS/MS analysis and visualized using MALDI-MSI. Spinalis muscle had the highest amount of identified PI molecular species, followed by loin, transparent tissue, and intermuscular fat tissue. The diacyl-PI molecular species containing hexadecadienoic, oleic, linoleic and eicosadienoic acids at the sn-2 position were mainly abundant in the loin and spinalis muscle, whereas those containing mead, arachidonic, docosatetraenoic, and docosapentaenoic acids at the sn-2 position were mainly abundant in both muscles as well as transparent tissues. Notably, the balance of PI molecular species differed among the tissues depending on fatty acid compositions at the sn-2 position. These results suggested that MALDI-MSI is a promising tool for assessing the association between individual pork tissues and the protective effects of PI molecular species against diseases related to pregnancy and cancer. To the best of our knowledge, this is the first report showing tissue-specific distributions of PI molecular species in pork chop using MALDI-MSI.

Unique Distribution of Diacyl-, Alkylacyl-, and Alkenylacyl-Phosphatidylcholine Species Visualized in Pork Chop Tissues by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging.

Phosphatidylcholine (PC) is the major phospholipid in meat and influences meat qualities, such as healthiness. PC is classified into three groups based on the bond at the sn-1 position: Diacyl, alkylacyl, and alkenylacyl. To investigate their composition and distribution in pork tissues, including longissimus thoracis et lumborum (loin) spinalis muscles, intermuscular fat, and transparent tissues, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Eleven diacyl-, seven alkylacyl-, and six alkenylacyl-PCs were identified using liquid chromatography (LC)-tandem MS (MS/MS) analysis. Despite many alkylacyl- and alkenylacyl-PC species sharing identical m/z values, we were able to visualize these PC species using MALDI-MSI. Diacyl- and alkylacyl- and/or alkenylacyl-PC species showed unique distribution patterns in the tissues, suggesting that their distribution patterns were dependent on their fatty acid compositions. PCs are a major dietary source of choline in meat, and the amount was significantly higher in the muscle tissues. Consumption of choline mitigates age-related memory decline and neurodegenerative diseases; therefore, the consumption of pork muscle tissues could help to mitigate these diseases. These results support the use of MALDI-MSI analysis for assessing the association between PC species and the quality parameters of meat.

Tissue-Specific Distribution of Sphingomyelin Species in Pork Chop Revealed by Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry.

Sphingomyelin (SM) species are major sphingolipids in pork meat that affect quality parameters, such as health benefits due to their protective properties against chronic diseases; however, their spatial distribution remains unclear. We used matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS) to investigate the distribution and composition of SM species in pork chop consisting of longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle. Four SM species were identified by liquid chromatography-electrospray ionization-tandem MS (MS/MS) and MALDI-MS/MS and visualized using MALDI-IMS. SM species containing stearic acid were predominantly distributed in the loin and spinalis muscle, whereas SM species containing palmitic, lignoceric, and nervonic acids were predominantly distributed in transparent tissue. These results indicated that the distribution of SM species differed among the pork tissues, depending on the tissue-specific fatty acid composition. The total amount including all identified SM species was higher in the loin than in spinalis muscle. Pork is reportedly associated with increased risk for chronic diseases due to the high amount of heme iron. From the observation of color, the amount of heme iron was lower in loin than in spinalis muscle. Thus, the degree of risk for chronic diseases might be lower in the loin than in spinalis muscle. This is the first report on the tissue-specific distribution of SM species in meat at a microscopic resolution using IMS. MALDI-IMS analysis may be useful in assessing the association between SM species and quality parameters of pork meat. PRACTICAL APPLICATION: Sphingomyelin (SM) species are major sphingolipids in pork meat. SM species affect quality parameters such as health benefits due to their protective properties against colon cancer and atherosclerosis. Matrix-assisted laser desorption/ionization-imaging mass spectrometry analysis combined with liquid chromatography-electrospray ionization-tandem mass spectrometry is a suitable method to directly investigate the distribution and composition of SM species at microscopic level among different tissues of pork meat. Therefore, this method is useful to assess the SM species-induced health effect of different tissues of pork meat.

Distribution of Flavan-3-ol Species in Ripe Strawberry Fruit Revealed by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging.

Flavan-3-ols, which comprise proanthocyanidins and their monomers, are major flavonoids in strawberries, and they have a wide range of biological activities and health benefits. However, their spatial distribution in strawberry fruit remains poorly understood. Therefore, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI), to visualize flavan-3-ols in ripe strawberry fruit. Peaks matching the m/z values of flavan-3-ols [M - H]- ions were detected in the negative ion mode using 1,5-diaminonaphthalene as matrix. Catechin and/or epicatechin, three B-type procyanidins, and two B-type propelargonidins were identified by MALDI-tandem MS. These flavan-3-ols were mainly distributed in the calyx, in and around the vascular bundles, and in the skin. In-source fragmentation of proanthocyanidins was determined using their standards, suggesting their distribution was mixed ion images of themselves, and fragment ions generated from those had a higher degree of polymerization. B-type procyanidins were predominantly distributed in the vascular bundles than in the skin, whereas B-type propelargonidins were almost equally distributed between the vascular bundles and skin, suggesting that their distribution patterns are different from the type of their flavan-3-ol monomers. Flavan-3-ols, especially B-type procyanidins, may help prevent pathogen infection not only in the skin but also in and around the vascular bundles.

Ulcer disease prophylaxis in koi carp by bath immersion with chicken egg yolk containing anti-Aeromonas salmonicida IgY.

Ulcer disease, caused by atypical Aeromonas salmonicida, is a serious concern in ornamental koi carp, because it induces skin ulceration, disfiguring ornamental fish and causing economic loses. The present study aimed to establish a novel prophylaxis with chicken egg yolk immunoglobulin, IgY, against ulcer disease and to assess its feasibility in the ornamental fish industry. Addition of egg yolk powder containing anti-A. salmonicida IgY to rearing water provided significant protection against an A. salmonicida bath infection, whereas administration of non-specific IgY did not. Consecutive immersion of fish into rearing water containing specific IgY completely prevented ulcer disease resulting from cohabitation infection, indicating that this prophylaxis could prevent infection from such type of contact. Thus, passive immunization induced by immersing fish into aquarium water containing specific IgY is a prospective prophylaxis against diseases caused by pathogens that invade the skin and gills.

Stimulating macrophage activity in mice and humans by oral administration of quillaja saponin.

Quillaja saponin (QS) was examined for its immunostimulating effect on mice and humans after oral administration. Mice fed QS for 24 h significantly increased in chemotactic and phagocytosis activities of peritoneal macrophages. This enhancing effect in both activities continued for 4-d after QS administration. Mice fed QS for 24 h prior to an interperitoneal challenge with Escherichia coli showed a higher survival rate than the control group. Peripheral blood analysis of volunteers showed significant increases in chemotactic and phagocytosis activities after oral administration of QS for 7 d. Furthermore, the volunteers did not show significant changes in immunoglobulin, transaminase, IL-1α, or TNF-α levels, or in serum albumin concentrations. Thus orally administered QS can effectively enhance the immune response through stimulation of macrophages without adverse effects.

Egg yolk-derived immunoglobulin (IgY) against Porphyromonas gingivalis 40-kDa outer membrane protein inhibits coaggregation activity.

The anaerobic bacterium Porphyromonas gingivalis, a major pathogen in periodontitis, aggregates with a number of oral bacteria to form dental plaque, which is important for its colonization. We previously cloned the gene coding the 40-kDa outer membrane protein (OMP) of P. gingivalis 381 and produced large amounts of the recombinant (r) protein. Affinity-purified rabbit antiserum against r40-kDa OMP effectively inhibited the coaggregation activity of P. gingivalis to oral bacteria, thus 40-kDa OMP was thought to be an important coaggregation factor of P. gingivalis. Further, since it is conserved among many P. gingivalis strains, this coaggregation factor may be an effective target for passive immunotherapy against P. gingivalis infection. Recently, passive immunization approaches using a specific antibody produced from hen egg yolk (IgY) have been developed for oral infectious diseases, and shown to be convenient and economic. In the present study, we immunized hens intramuscularly with r40-kDa OMP and obtained highly purified IgY from the egg yolks. The purified IgY specifically recognized r40-kDa OMP and also reacted with a functional coaggregation-associated domain peptide of 40-kDa OMP. Our results demonstrated that a ratio of purified IgY as low as 2.5 microg/150 microl significantly inhibited the coaggregation of P. gingivalis with Streptococcus gordonii, which was verified by a visual coaggregation assay and radioactivity-based quantitative micro-coaggregation assay. We concluded anti-r40-kDa OMP IgY may be useful for passive immunization against periodontal diseases caused by P. gingivalis infection.

Inhibition of Porphyromonas gingivalis hemagglutinating activity by IgY against a truncated HagA.

Porphyromonas gingivalis has been implicated as an important pathogen in the development of periodontitis. Hemagglutinins have been identified as important adhesion molecules, allowing Porphyromonas gingivalis to adhere to gingival tissue cells, and to attach and lyse erythrocytes in order to uptake Fe ions as essential nutrition. One hemagglutinin, hemagglutinin A (HagA), has been molecularly cloned via functional screening for hemagglutinating activity. We previously cloned the gene encoding the 200-kDa cell-surface antigenic protein that was reacted by sera from periodontitis patients and was identified as a truncated protein of HagA by nucleotide sequence analysis. We further subcloned the gene encoding an 122-kDa protein (122k-HagA) which is a fusion protein composed of an 80-kDa truncated HagA containing the functional motif PVQNLT and a 42-kDa maltose binding protein. Passive immunization against infectious pathogens by specific antibodies produced from hen egg yolk antibody (IgY) has been extensively developed. In the present study, to develop passive immunotherapy against periodontal disease, we purified the recombinant 122k-HagA and used this to immunize hens and produce IgY. The purified IgY reacted with the recombinant 122k-HagA and the synthetic peptide containing PVQNLT, and inhibited hemagglutinating activity of Porphyromonas gingivalis. Thus, the novel IgY may be useful in the development of a passive immunization against periodontal diseases caused by P. gingivalis infection.

Production of rabies neutralizing antibody in hen's eggs using a part of the G protein expressed in Escherichia coli.

In an attempt to produce anti-rabies immunoglobulin affordable for people living in developing countries, we have immunized layer chickens with a part of the G protein of rabies virus expressed in Escherichia coli. Immunoglobulin (IgY) was purified from the yolks of eggs layed by immunized hens. It was revealed in vitro that the antibody specifically bound to virions as well as cells infected with rabies virus. Moreover, the antibody apparently neutralized rabies virus infectivity. Inoculation of the antibody into mice infected with rabies virus reduced the mortality caused by the virus, suggesting that IgY directed to the part of the G protein expressed in E. coli could serve as a possible alternative to currently available anti-rabies human or equine immunoglobulins.

Detection of rabies-specific antigens by egg yolk antibody (IgY) to the recombinant rabies virus proteins produced in Escherichia coli.

We obtained rabies-specific egg yolk antibodies (IgY) by immunizing hens with recombinant His-tagged nucleoprotein and phosphoprotein (rN, rP) of the rabies virus (CVS-11 strain) expressed in Escherichia coli. The anti-rN and rP IgY were shown to bind specifically to the respective proteins of the CVS-11 strain of rabies virus by Western blotting, immune fluorescent assay and immunohistochemistry, indicating that IgY to rabies recombinant proteins could serve as a reagent for diagnosis of rabies virus infection.

Improvement of functional properties of egg white protein through phosphorylation by dry-heating in the presence of pyrophosphate.

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 3.0-7.0 and 85 degrees C for 1 and 5 days, and the functional properties of the phosphorylated EWP (PP-EWP) were investigated. The phosphorylation was accelerated with a decrease of pH from 7.0 to 3.0 and for heating times from 1 to 5 days. The phosphorus content of EWP increased approximately 1.05% by dry-heating at pH 4.0 and 85 degrees C for 5 days in the presence of pyrophosphate, which was higher than that of casein. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The surface hydrophobicity of EWP increased by phosphorylation. The heat stability, emulsifying properties, and digestibility of EWP were improved by phosphorylation. The calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation. A firmer and transparent heat-induced gel of PP-EWP was obtained, and the water-holding capacity of heat-induced PP-EWP gel was higher that that of the control. These results suggest that phosphorylation by dry-heating in the presence of pyrophosphate is a useful method for improving the functional properties of EWP.