Yolk Immunoglobulin Research Articles

To Reduce the Effects of Experimental Aflatoxicosis in Broiler Chicks Using Specific Egg Yolk Immunoglobulin (IgY)

Background : This experiment was conducted to evaluate the effects of the specific egg yolk immunoglobulin (IgY) on reducing the defects of experimental aflatoxicosis in broilers. Materials and Methods : In a completely randomized design, a total of 128 Ross 308 broiler chicks were used in 4 treatments, 4 replicates and 8 observations (chicks) for 42 days. Treatments were: 1) control; 2) diet containing 1 ppm aflatoxin B1; 3) diet contaminated with 1 ppm aflatoxin B1 + 0.75 % of egg yolk containing IgY; 4) diet contaminated with 1 ppm aflatoxin B1 + 1.5 % of egg yolk containing IgY. Results : The results showed that the use of aflatoxin containing diet significantly increased the feed conversion ratio, serum cholesterol concentration, serum gamma-glutamyl transferase (GGT), and aspartate aminotransferase (AST). Also, experimental aflatoxicosis resulted in reduced feed intake, weight gain, serum total protein and albumin concentrations (p<0.05); as well as the liver histopathologic lesions. IgY containing egg yolk (0.75% and 1.5%) added to the contaminated ration improved feed intake, weight gain and feed conversion ratio compared to treatment 2 (p<0.05). Compared to treatment 2, serum cholesterol concentration decreased while total protein concentration increased in treatments 3 and 4 (p<0.05). Liver tissue was approximately normal with mild effects on hepatocytes and mild cytoplasmic changes in chicks receiving treatments 3 and 4. Conclusion : It can be concluded that specific IgY is effective in reducing the defects of experimental aflatoxicosis as well as improving performance in broilers.

Production of anti-Trichophyton rubrum egg yolk immunoglobulin and its therapeutic potential for treating dermatophytosis

The aim of this study was to estimate the therapeutic potential of specific egg yolk immunoglobulin (IgY) on dermatophytosis caused by Trichophyton rubrum. The IgY was produced by immunizing hens with cell wall proteins of T. rubrum, extracted from eggs by PEG precipitation and then purified by ammonium sulfate precipitation. The cross-reactivity (CR) with other fungi, growth inhibition on T. rubrum in vitro and therapeutic effect on T. rubrum infection in BALB/C mice of the specific IgY were then evaluated. Anti- T. rubrum cell wall proteins IgY (anti-trCWP IgY) presented a certain degree of cross-reactivity with different fungi. In the in vitro and in vivo activity researches, Anti-trCWP IgY showed a significant dose-dependent growth inhibitory effect on T. rubrum in vitro and a significant dose-dependent therapeutic effect on T. rubrum infection in BALB/C mice.

Effect of an egg yolk immunoglobulin（IgY）product on oocyst shedding and blood and fecal IgY concentrations in &lt;i&gt;cryptosporidium&lt;/i&gt;-infected calves

Effect of an egg yolk immunoglobulin（IgY）product on oocyst shedding and blood and fecal IgY concentrations in &lt;i&gt;cryptosporidium&lt;/i&gt;-infected calves

Colorimetric detection of Staphylococcus aureus using gold nanorods labeled with yolk immunoglobulin and urease, magnetic beads, and a phenolphthalein impregnated test paper.

A colorimetric test is described for the rapid detection of Staphylococcus aureus (SA). Gold nanorods (AuNRs) are first labeled with urease and yolk immunoglobulin (IgY). This probe can specifically bind SA. In the next step, nonspecific magnetic beads and sample are added. This leads to the formation of the AuNR-IgY-SA-nMB immunocomplex which is then magnetically separated. Finally, a solution of urea is added to the supernatant. Ureases catalyzes the decomposition of urea which results in an increase in the pH value. The increase in the pH value is detected by using a phenolphthalein test paper which undergoes a color change from white to pink. The analytical process can be completed within 20min. The method is highly specific and can detect as little as 476cfu·mL-1. It was verified by analyzing contaminated Chinese cabbage and beef samples, and 1000cfu·mL-1 of SA were accurately detected. Graphical abstract Schematic representation of a colorimetric method for the detection of Staphylococcus aureus based on the immunocomplex formed from dual-labeled gold nanorod (AuNR) probe, bacteria and non-specific magnetic bead (nMB). This method can be completed within 20min.

Protective effect of chicken egg yolk immunoglobulins (IgY) against enterotoxigenic Escherichia coli K88 adhesion in weaned piglets

BackgroundEnterotoxigenic Escherichia coli K88 (E. coli K88) are considered as a major cause of diarrhea and death in newly weaned piglets. Oral passive immunization with chicken egg yolk immunoglobulins (IgY) have attracted considerable attention for treatment of gastrointestinal infection due to its high specificity. In this study it was estimated the protective effect of anti-K88 fimbriae IgY against E. coli K88 adhesion to piglet intestinal mucus in vitro and to investigate the potential use of IgY for controlling E. coli-induced diarrhea in weaned piglets in vivo.ResultsE. coli K88 was incubated with IgY for 24 h, and the bacterial growth profiles showed that specific IgY with a concentration higher than 5 mg/mL was observed to significantly inhibit the growth of E. coli K88 compared to nonspecific yolk powder in a liquid medium. Moreover, pretreatment with 50 mg/mL of IgY was found to significantly decrease the adhesion ability of E. coli K88 to porcine jejunal and ileal mucus, further supported by the observations from our immunofluorescence microscopic analysis. In vivo, administration of IgY successfully protected piglets from diarrhea caused by E. coli K88 challenge. Additionally, IgY treatment efficiently alleviated E. coli-induced intestinal inflammation in piglets as the gene expression levels of inflammatory cytokines TNF-α, IL-22, IL-6 and IL-1β in IgY-treated piglets remained unchanged after E. coli K88 infection. Furthermore, IgY significantly prevented E. coli K88 adhering to the jejunal and ileal mucosa of piglets with E. coli infection and significantly decreased E. coli and enterotoxin expression in colonic contents.ConclusionOutcome of the study demonstrated that IgY against the fimbrial antigen K88 was able to significantly inhibit the growth of E. coli K88, block the binding of E. coli to small intestinal mucus, and protect piglets from E. coli-induced diarrhea. These results indicate that passive immunization with IgY may be useful to prevent bacterial colonization and to control enteric diseases due to E. coli infection. The study has great clinical implication to provide alternative therapy to antibiotics in E coli induced diarrhea.

Meat Quality on Sentul Cocks with Different Immunoglobulin Yolk Concentrations

&lt;p class="abstrak2"&gt;Sentul cocks is one of the native chicken breeds in Indonesia which is originally raised by Villagers in Ciamis District, West Java. Healthy chicken cells can function properly, especially in the metabolic process. Healthy chickens are expected to produce better muscle development. IgY is a protein molecule substance that can neutralize a number of microorganisms that cause infection. The purpose of this study was to evaluate the effect of IgY concentration on physicochemical and organoleptic qualities of meat. This study used 20 cocks, 4th month ages, consist of 2 treatments (IgY concentrations above 9.30 ± 0.45 mg mL&lt;sup&gt;-1&lt;/sup&gt; and IgY concentrations below 9.30 ± 0.45 mg mL&lt;sup&gt;-1&lt;/sup&gt;). The variable observed include physicochemical and organoleptic quality of meat. The study was used completely randomized design. Data were analyzed by t-test. The result concluded that cocks with concentrations above 9.30 ± 0.45 mg mL&lt;sup&gt;-1&lt;/sup&gt; produced meat with lower malonaldehyde. The low content of malonaldehyde in meat shows that the meat produced is healthier.&lt;/p&gt;

Effect of chicken egg yolk immunoglobulins on serum biochemical profiles and intestinal bacterial populations in early-weaned piglets.

This study was conducted to test the hypothesis that dietary supplementation with anti‐E. coli, chicken egg yolk immunoglobulins (IgY), may affect early weaned piglet (EWP) intestinal functions and enteric micro‐organisms. One hundred and forty‐eight ([Landrace × Yorkshire] × Duroc) piglets, weaned at age day 21, were randomly assigned to receive one of three diets for 14 days. Treatment group one (control group) was fed the base diet. Treatment group two (antibiotics group) was fed the base diet which was supplemented with 100 ppm colistin sulphate and 15 ppm enramycin; treatment group three (IgY group) was fed the base diet which was supplemented with 500 mg/kg anti‐E. coli IgY. The study evaluated the effects on EWPs of IgY on growth, serum biochemical, inflammatory profiles and also digestion content intestinal bacterial populations. Results showed no significant difference in diarrhoea rates between IgY‐fed EWPs and antibiotic‐treated EWPs. Serum biochemical analysis showed that EWPs fed an IgY‐containing diet had both lower (p < 0.05) cholesterol and low‐density lipoprotein compared to antibiotic‐treated EWPs. Escherichia coli populations measured in IgY‐fed EWP ileal contents, compared to the control group, were significantly reduced (p < 0.05). Enterococcus, Lactobacillus, Clostridium and Bifidobacterium populations were unaffected by the IgY treatment. Larger (p < 0.05) Enterococcus populations and lower (p < 0.05) expression levels of heat‐stable enterotoxin b (STb) were observed in IgY‐fed EWP caecal digesta compared to the control group. Enteric Lactobacillus significantly decreased (p < 0.05) in EWPs fed antibiotics while it was unaffected by IgY treatment. Dietary supplementation with anti‐E. coli IgY has the potential to suppress enteric E. coli growth, but not Lactobacillus, Clostridium and Bifidobacterium. This promotes and maintains a healthy EWP intestinal environment. These findings suggest that IgY may be used as an alternative to antibiotics in EWP diets.

Effect of egg yolk immunoglobulin on an experimental periodontitis model induced by Porphyromonas gingivalis

Effect of egg yolk immunoglobulin on an experimental periodontitis model induced by Porphyromonas gingivalis

Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies.

Bacterial meningitis is a severe disease that is fatal to one-third of patients. The major cause of meningitis in neonates is Escherichia coli (E. coli) K1. This bacterium synthesizes an outer membrane protein A (OmpA) that is responsible for the adhesion to (and invasion of) endothelial cells. Thus, the OmpA protein represents a potential target for developing diagnostic and therapeutic agents for meningitis. In this study, we expressed recombinant OmpA proteins with various molecular weights in E. coli. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the molecular size of OmpA's full length (FL) and truncated proteins. OmpA-FL protein was purified for immunizing chickens to produce immunoglobulin yolk (IgY) antibodies. We applied phage display technology to construct antibody libraries (OmpA-FL scFv-S 1.1 × 107 and OmpA-FL scFv-L 5.01 × 106) to select specific anti-OmpA-FL scFv antibodies; these were characterized by their binding ability to recombinant or endogenous OmpA using ELISA, immunofluorescent staining, and confirmed with immunoblotting. We found 12 monoclonal antibodies that react to OmpA fragments; seven scFvs recognize fragments spanning amino acid (aa) residues 1-346, aa 1-287, aa 1-167, and aa 60-192, while five scFvs recognize fragments spanning aa 1-346 and aa 1-287 only. Two fragments (aa 246-346 and aa 287-346) were not recognized with any of the 12 scFvs. Together, the data suggest three antigenic epitopes (60 aa-160 aa, 161 aa-167 aa, 193 aa-245 aa) recognized by monoclonal antibodies. These scFv antibodies show strong reactivity against OmpA proteins. We believe that antibodies show promising diagnostic agents for E. coli K1 meningitis.

Enterovirus type 71-immunized chicken egg yolk immunoglobulin has cross antiviral activity against coxsackievirus A16 in vitro.

To exploit a cross passive immunotherapy for enterovirus-induced hand-foot-and-mouth disease (HFMD), the cross antiviral activity of a neutralizing antibody against enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) was investigated in vitro. White Leghorn specific-pathogen-free chickens were immunized with EV71 antigens and a specific isolated immunoglobulin (IgY) was prepared from the chicken egg yolk. IgY was further purified and characterized by SDS-PAGE, ELISA, western blotting and bidirectional immune agar diffusion testing. The antiviral activity and dose-response of the IgY were determined by assessing the cytopathic effect in rhabdomyosarcoma (RD) cells in vitro. It was indicated that the levels of IgY were increased at day 7, peaked at week 7 and were maintained at a higher level for 4 weeks following immunization when compared with the negative control. The results of western blotting and bidirectional immune agar diffusion testing revealed that the IgY had cross-binding properties in EV71 and CVA16 strains through targeting the envelope proteins (VP0, VP1 and VP3) of EV71 and CVA16. Neutralization assay results indicated that the infectivity of EV71 and CVA16 strains in RD cells was cross-blocked by IgY in a dose-dependent manner. To conclude, these findings indicate that IgY has cross antiviral activity against EV71 and CVA16 in vitro, and could potentially be developed as a passive immunotherapy for EV71- and CVA16-induced HFMD.

Preparation and identification of chicken egg yolk immunoglobulins against human enterovirus 71 for diagnosis of hand-foot-and-mouth disease

Human enterovirus 71 (EV71) is one of the major pathogens that causes hand-foot-and mouth disease, and there is an urgent need for rapid diagnosis of EV71 virus infection for early antiviral treatment. The aim of this study was to prepare chicken egg yolk immunoglobulin Y (IgY) for the diagnosis of enterovirus type 71 infection. The antibodies were raised by intramuscular immunization of laying hens with inactivated human EV71 and isolated from the egg yolk by multiple steps of polyethylene glycol 6000 extraction. The average concentration of IgY antibody was 26.60 mg/mL during the whole immunization. After the first immunization, the IgY titer gradually increased, and reached the peak on the 55th days. Meanwhile, the use of western blotting test demonstrated that specific IgY binds specifically to capsid proteins VP2 and VP3 of EV71 virus. Furthermore, a facile one-step method based on turn-on fluorescence sensing was developed by using IgY antibodies, which can detect EV71 virus at low concentrations of 104 PFU/mL and was 94.44% coincidence with RT-PCR in 30 clinical samples. These findings indicate that EV71-IgY antibodies are an easily prepared and rich source of antibodies that offers a potential alternative strategy for routine screening of EV71 infection.

The preparation and antibacterial effect of egg yolk immunoglobulin (IgY) against the outer membrane proteins of Vibrio parahaemolyticus

Vibrio parahaemolyticus causes not only various diseases in aquaculture animals but also seafood-borne illness in humans. Outer membrane proteins (OMPs) are species-specific proteins found in bilayer membranes of gram-negative bacteria. Egg yolk immunoglobulin (IgY) has been reported to serve as oral administration of antibodies against bacteria and virus. The present research extracted and identified OMPs from V. parahaemolyticus, and then the extracted OMPs were used to immunize hens to obtain specific IgY. The efficacy of IgY against V. parahaemolyticus were investigated in vitro and in vivo. The specific IgY effectively inhibited the growth of V. parahaemolyticus in liquid medium rather than Escherichia coli and Staphylococcus aureus. Specific IgY antibodies were incorporated into extruded food pellets and fed to bacteria-challenged white pacific shrimp to observe the anti-bacterial effect in vivo. The bacterial loads in muscles of V. parahaemolyticus infected shrimp fed with specific IgY-included diets were significantly fewer than those fed with non-specific IgY-included diets. The superoxide dismutase activities in muscles of infected shrimp fed with specific IgY-included diets were significantly higher than the control group. The results suggested that the specific IgY effectively inhibited the growth of V. parahaemolyticus and introduced passive immunity to shrimp. © 2018 Society of Chemical Industry.

Enterobacter cloacae associated with mass mortality in zoea of giant freshwater prawns Macrobrachium rosenbergii and control with specific chicken egg yolk immunoglobulins (IgY)

Outbreaks of mass mortality among cultured zoea of M. rosenbergii occurred in different hatcheries of Gaoyou country of China in April 2017. Of the isolates obtained from the diseased zoea, XL3–1 was pathogenic to the zoea and postlarva of M. rosenbergii in the virulence test, with a LD50 value 3.2 × 106 CFU/mL and 3.5 × 106 CFU/mL, respectively. XL3–1 was identified as Enterobacter cloacae by biochemical characteristics and molecular tools. Detection of virulence-associated genes by PCR indicated that XL3–1 was positive for irp2, fhuA, sodB, sltA, flaD and OmpX. According to the antibiotic resistant assay, the isolate was sensitive to quinolones, while they were resistant to cephalosporins and penicillins. This study also investigated the protection of specific chicken egg yolk antibodies (IgY) in E. cloacae-infected M. rosenbergii. The highest titer against E. cloacae of the obtained specific IgY by ELISA was 1: 213. M. rosenbergii treated with specific egg yolk powder could achieve the survival rate of 67% after E. cloacae infection, while M. rosenbergii treated with non-specific egg yolk powder or no egg yolk powder could achieve survival rates of 20% and 17%, respectively. As well, the bacterial burden in hepatopancreas was significantly lower in M. rosenbergii treated with specific egg yolk powder than those treated with non-specific egg yolk powder. This study clarified the virulence characteristics and antibiotic resistance of E. cloacae, and provided a strategy to control the disease by using specific IgY.

Egg Yolk Immunoglobulin Supplementation Prevents Rat Liver from Aflatoxin B1-Induced Oxidative Damage and Genotoxicity.

Egg yolk immunoglobulins (IgY), as nutraceutical supplement for therapeutic or prophylactic intervention, have been extensively studied. The effects of IgY on small molecular toxin-induced toxicity in animals are unclear. In the present study, the protection of highly purified and specific anti-AFB1 IgY against AFB1-induced genotoxicity and oxidative damage on the rat liver model were investigated. Our results revealed that AFB1 induced significant oxidative damage markers, as well as AFB1-induced protein expression in antioxidant, pro- and antiapoptosis processes in rat liver. These effects could be significantly inhibited by cogavage with anti-AFB1 IgY in a dose-dependent manner. However, anti-AFB1 IgY did not significantly induce hepatic CAT and SOD1. To explore mechanisms, metabolite experiments were established to evaluate the influence of anti-AFB1 IgY on the absorption of AFB1 in rats. Middle and high doses of anti-AFB1 IgY reduced hepatic AFB1-DNA adducts by 43.3% and 52.9%, AFB1- N7-guanine urinary adducts by 19.6% and 34.4%, and AFB1-albumin adducts by 10.5% and 21.1%, respectively. The feces of high dose anti-AFB1 IgY cogavaged rats contained approximately 2-fold higher AFB1 equivalents at 3-6 h after ingestion than AFB1 group feces, indicating IgY inhibited AFB1 uptake. These results had provided insight that anti-AFB1 IgY could prevent animal organs from damage caused by AFB1 and will be beneficial for the application of detoxification antibody as a supplement in food.

Egg yolk immunoglobulin interactions with Porphyromonas gingivalis to impact periodontal inflammation and halitosis

Porphyromonas gingivalis is a pathogenic Gram-negative anaerobic bacterium that colonizes the subgingival region of gums. These bacteria can invade periodontal tissues, form plaques, and produce volatile sulfur compounds (VSC) and volatile organic compounds (VOC). Egg yolk immunoglobulin (IgY) that was specifically produced in egg yolks after chickens were challenged with P. gingivalis could control and prevent oral diseases caused by P. gingivalis. The releases of P. gingivalis offensive metabolic odors in vitro and in vivo were determined using a Halimeter and GCMS. With IgY bacterial growth was inhibited, and the relative amounts of VOC and VSC were decreased. The scores for the oral health index and the levels of IL-6 and TNF-α are also decreased. All treatment groups showed significant anti-inflammatory effects, which strongly suggests that specifically IgY against P. gingivalis may be an effective treatment for the prevention and protection of periodontal inflammation and halitosis.

Egg yolk immunoglobulins’ impact on experimental periodontitis caused by Porphyromonas gingivalis

Periodontitis is a chronic inflammatory disease caused by Porphyromonas gingivalis that leads to a series of periodontal tissue injuries. Egg yolk immunoglobulins (IgY) is procuded in egg yolk and inhibits P. gingivalis. The aim was to evaluate the effect of IgY on experimental periodontitis caused by P. gingivalis. The second molars of rats were ligatured using medical 5-0 silk and smeared with P. gingivalis to induce experimental periodontitis. Then, the rats were smeared with 2mL IgY solutions or 0.9% NaCl in the oral cavity for up to 4wk. The scores for gingival index, plaque index and probe on bleeding, the levels of IL-6 and TNF-α, X-ray radiography and histology were used to determine the efficacy of the IgY on experimental periodontitis. The clinical indices improved; the levels of IL-6 and TNF-α were significantly (p< 0.05) decreased; the X-rays and histomorphological observations suggested that the periodontal inflammation and periodontitis were alleviated compared to the control. IgY showed significant effects on anti-inflammatory, anti-coaggregation activity, and protected against alveolar bone loss. Therefore, it had a beneficial effect on preventing experimental periodontitis caused by P. gingivalis.

Evaluation of lozenges containing egg yolk antibody against Porphyromonas gingivalis gingipains as an adjunct to conventional non-surgical therapy in periodontitis patients: A randomized controlled clinical trial.

In a previous pilot study, one-time application of anti-Porphyromonas gingivalis gingipain egg yolk immunoglobulin (IgY) into scaling and root planing (SRP)-treated periodontal pockets showed profound improvement of clinical and bacteriological parameters in patients with chronic periodontitis. The present study aims to evaluate the efficacy of daily use of lozenges fortified with the antibody as an adjunct to non-surgical therapy in patients with periodontitis. Sixty-four patients with periodontitis were divided randomly into a test and a placebo group. The groups were treated by SRP followed by a daily use of lozenges containing either specific IgY against P. gingivalis gingipains (test) or a sham-immune IgY (placebo). Gingival bleeding index (GBI), probing pocket depth (PD) and quantitation of P. gingivalis in the gingival pockets were assessed at baseline and 8weeks after the initiation of treatment and compared by using Wilcoxon signed rank test, Mann-Whitney U-test or t test. Both groups showed significant improvement of all parameters at 8weeks post treatment (P<0.001). There was a significant difference in reduction of GBI (P<0.001) and P. gingivalis cell counts (P<0.05) in the test group compared with the placebo group. The reduction of PD was greater in the test group compared with the placebo group although there was no statistically significant difference between the two groups. The adjunctive use of lozenges containing IgY antibody against gingipains from P. gingivalis resulted in clinical and microbiological benefits in the treatment for chronic periodontitis. Additional investigations are needed to examine if the IgY brings benefits to case patients who do not receive SRP.

Production and application of anti-nucleoprotein IgY antibodies for influenza A virus detection in swine

Influenza A virus (IAV) causes an important respiratory disease in mammals and birds leading to concerns in animal production industry and public health. Usually, antibodies produced in mammals are employed in diagnostic tests. However, due to animal welfare concerns, technical advantages and the high cost of production, alternatives to the production of antibodies in mammals have been investigated. The aim of this study was to produce egg yolk immunoglobulin (IgY) in laying hens against a highly conserved protein (nucleoprotein- NP) of IAV and to evaluate the application of anti-NP IgY antibodies in virus detection by immunocytochemistry (ICC) and immunohistochemistry (IHC). Three laying hens of the White Leghorn line were inoculated seven times with a recombinant NP protein and their eggs collected seven days after the 3rd, 5th and 7th inoculations. Immunoglobulin Y antibodies were purified from egg yolk through precipitation with ammonium sulfate. The titers and specificity of the purified antibodies were determined by ELISA, western blotting, ICC and IHC. High levels of specific anti-NP antibodies were detected by ELISA after the 5th inoculation, reaching a peak after the 7th inoculation. The mean yield of total protein in yolk after the 7th inoculation was 13.5 mg/mL. The use of western blotting and ICC demonstrated that anti-NP IgY binds specifically to NP protein. Moreover, the use of anti-NP IgY antibody in ICC test revealed positive staining of MDCK cells infected with IAV of the three subtypes circulating in swine (H1N1, H1N2, and H3N2). However, no staining was observed in lung tissues through the IHC test. The data obtained showed that anti-NP IgY antibodies bound specifically to influenza virus NP protein, detecting the main virus subtypes circulating in swine, reinforcing their usefulness in the influenza diagnosis.

Features of chicken egg yolk immunoglobulin (IgY) against the infection of red-spotted grouper nervous necrosis virus

Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most important viruses which mainly infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide. Currently, there are no effective vaccines available for the fish larva infected with NNV. Immunoglobulin yolk (IgY) origin of oviparous animals is passed from the blood serum and concentrated in the egg yolk. With the advantages of high yield, cost-effectiveness, and high stability, IgY can be widely used in passive immunization, especially in young animals in which adaptive immunity is not fully developed. In this study, we have cloned and expressed the recombinant capsid protein of RGNNV in Escherichia coli and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens. Water-soluble fractions (WSF) of the specific IgY were isolated from egg yolk and purified by two-step precipitation with saturated ammonium sulfate salting. By Enzyme linked immunosorbent assay (ELISA), the titer of the IgY reached a peak at the 6th week post of immunization and had a strong stability at a wide range of temperature, pH, and pepsin enzyme digestion. The purified IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line (GF-1), indicating that it was highly specific and effectively recognized RGNNV. The results will pave a new way for the prevention of RGNNV infection.