Development and evaluation of an immunoassay for the quantification of N-acetylneuraminic acid (Neu5Ac) in foods and biosamples.

N-acetylneuraminic acid is an active ingredient in tonic foods and an important additive in foods and biopharmaceuticals. To address the limitations of existing methods of N-acetylneuraminic acid quantification, we developed an immunoassay based on antibodies induced in hens using artificial antigen, showing high sensitivity and specificity with no cross-reactivity with eight N-acetylneuraminic acid analogues. An IgY-based indirect competitive enzyme-linked immunosorbent assay showed a detection range of 1.14 to 70.08 ng/mL and a limit of detection of 0.57 ng/mL. In spiked samples, recoveries by the indirect competitive enzyme-linked immunosorbent assay ranged from 74.05% to 110.87% compared with HPLC (73.01% to 108.8%). Consistency between the indirect competitive enzyme-linked immunosorbent assay and HPLC was satisfactory (R2 = 0.9736), demonstrating this established immunoassay as a rapid and reliable approach for N-acetylneuraminic acid analysis. The assay described in this study provides an important method for the screening of N-acetylneuraminic acid in biological samples and foodstuffs.

Evaluation of specific chicken IgY antibody value developing diagnostic capture antibody ELISA kit against Foot and Mouth disease.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboard titration test was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications.

Broiler chicken distal jejunum microbial communities are more responsive to coccidiosis or necrotic enteritis challenge than dietary anti-interleukin-10 in a model using Salmonella Typhimurium- Eimeria maxima- Clostridium perfringens coinfection.

Dietary anti-interleukin (IL)-10 antibodies may protect broiler performance during coccidiosis by inhibiting Eimeria host-evasion pathways; however, anti-IL-10's effects on microbial communities during coccidiosis and secondary Clostridium perfringens (necrotic enteritis) challenge is unknown. The study objectives were to assess the jejunal microbiota of broilers fed anti-IL-10 during E. maxima ± C. perfringens challenge. Two replicate studies using Ross 308 chicks placed in wire-floor cages (32 cages/ replicate study; 20 chicks/ cage) were conducted, with chicks assigned to diets ± 0.03% anti-IL-10 for 25 d. In both replicate studies, challenge-designated chicks were inoculated with 1 × 108Salmonella Typhimurium colony forming units (CFU) at placement. On d14, S. Typhimurium-inoculated chicks were gavaged with 15,000 sporulated Eimeria maxima M6 oocysts and half the E. maxima-challenged chicks received 1×108C. perfringens CFUs on d 18 and 19. Six chicks/ treatment were euthanized for distal jejunum content collection at baseline (d 14), 7 d post-inoculation (pi) with E. maxima/ 3 dpi with C. perfringens (peak) or 11 dpi with E. maxima/ 7 dpi with C. perfringens (post-peak) for 16S rRNA gene amplicon sequencing. Sequences were quality screened (Mothur V.1.43.0) and clustered into de novo operation taxonomical units (OTU; 99% similarity) using the SILVA reference database (v138). Alpha diversity and log-transformed relative abundance data were analyzed in SAS 9.4 with replicate study, diet, challenge, and timepoint main effects plus associated interactions (P ≤ 0.05). Few baseline changes were observed, but E. maxima ± C. perfringens challenge reduced Romboutsia and Staphylococcus relative abundance 4- to 800-fold in both replicate studies (P ≤ 0.008). At peak challenge with secondary C. perfringens, feeding anti-IL-10 instead of the control diet reduced Clostridium sensu stricto 1 relative abundance 13- and 1,848-fold in both replicate studies (P < 0.0001); however, OTUs identified as C. perfringens were not affected by dietary anti-IL-10. These results indicate that anti-IL-10 does not affect the jejunal microbiota of unchallenged broilers, while coccidiosis or necrotic enteritis challenge generally contributed to greater microbiota alterations than diet.

Passive immunization of mice with IgY anti-H5N1 protects against experimental influenza virus infection and allows development of protective immunity.

Influenza virus contributes substantially to the global human and animal disease burden. To protect individuals against disease, strategies are needed to minimize the time an individual is at risk of developing disease symptoms. Passive immunization using avian IgY antibodies can protect individuals against a variety of pathogens, including influenza virus. Yet the effect of IgY administration on generation of protective immunity is largely unknown. To address the effect of passive immunization on the host immune response development, adult or aged, male and female C57BL/6NCrl mice received chicken IgY anti-H5N1, normal IgY or PBS intranasally four hours before, and 20 hours after intranasal infection with H1N1 influenza A virus (PR8). The mice receiving cross-reactive IgY anti-H5N1 were protected from disease and developed influenza virus-specific memory T cells similar to control-treated mice. When re-challenged with PR8 35 days post primary infection IgY anti-H5N1-treated mice were fully protected. Moreover, when challenged with heterologous H3N2 influenza A virus (X-31) or with PR8 three months post infection the mice were protected against severe disease and death, albeit a slight transient weight loss was noted. The results show that passive immunization with IgY anti-H5N1 is safe and protects mice against disease induced by influenza virus without inhibiting development of protective immunity after virus exposure. This indicate that passive immunization can be used as prophylactic therapy in combination with immunization to prevent disease.

Monoclonal IgY antibodies: advancements and limitations for immunodiagnosis and immunotherapy applications.

Due to their high specificity and scalability, Monoclonal IgY antibodies have emerged as a valuable alternative to traditional polyclonal IgY antibodies. This abstract provides an overview of the production and purification methods of monoclonal IgY antibodies, highlights their advantages over polyclonal IgY antibodies, and discusses their recent applications. Monoclonal recombinant IgY antibodies, in contrast to polyclonal IgY antibodies, offer several benefits. such as derived from a single B-cell clone, monoclonal antibodies exhibit superior specificity, ensuring consistent and reliable results. Furthermore, it explores the suitability of monoclonal IgY antibodies for low- and middle-income countries, considering their cost-effectiveness and accessibility. We also discussed future directions and challenges in using polyclonal IgY and monoclonal IgY antibodies. In conclusion, monoclonal IgY antibodies offer substantial advantages over polyclonal IgY antibodies regarding specificity, scalability, and consistent performance. Their recent applications in diagnostics, therapeutics, and research highlight their versatility.

Chicken egg yolk antibodies (IgY) and monoclonal antibodies: advancements and limitations for immunodiagnosis and immunotherapy applications Chicken egg yolk antibodies (IgY antibodies) and monoclonal antibodies (mAbs) are two types of antibodies used in medical applications. IgY antibodies are cost-effective, stable, and specific, with the advantage of not triggering harmful immune responses. However, they may have limitations in identifying certain target areas and availability. On the other hand, mAbs are highly specific and can detect multiple target areas on antigens, but their production is expensive and may cause immune responses. Despite these drawbacks, both IgY antibodies and mAbs show promise in various applications such as infectious disease diagnosis, cancer treatment, and autoimmune disorders. Ongoing developments in antibody technology are likely to expand their applications in immunology. This review provides an overview of the strengths and limitations of IgY antibodies and mAbs in immunodiagnosis and immunotherapy, as well as their role in pandemic control.

Generation of chicken-based IgY polyclonal antibodies against Dendroaspis polylepis and preclinical evaluation of envenomation-neutralizing efficacy vis-à-vis selected commercial antivenoms.

The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD50 of D. polylepis was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 µg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.

Development, Optimization and Evaluation of a Sensitive Enzyme-Linked Immunosorbent Assay (ELISA) Prototype for Detection of Chicken-Based IgY Polyclonal Antibodies against Toxins of D. polylepis Venom.

Life-threatening medical issues can result from snakebite, and hence this is a public health concern. In many tropical and subtropical nations such as Kenya, where a wide variety of poisonous snakes are prevalent, diagnosis of snakebite in health facilities is imperative. Different antivenoms are needed to treat the venom of different snake species. Nonetheless, it might be difficult for medical professionals to identify the exact snake species that envenomated a patient due to the similarities of several snake envenomations' clinical symptoms. Therefore, the necessity for an assay or technique for identifying venomous species is critical. The current study sought to develop a sensitive ELISA prototype for the detection of D. polylepis venom in Kenya using generated chicken-based IgY polyclonal antibodies. Serum samples containing specific chicken-based IgY antibodies previously raised against D. polylepis venom toxins were used in the assay development. ELISA parameters were optimized, and the developed assay was assessed for applicability. The limit of detection (LoD) of the ELISA for neurotoxic venoms was determined to be 0.01 µg/mL. Successful discrimination between neurotoxic and cytotoxic venoms was achieved by the ensuing inhibition ELISA assay. The developed assay showed the capability of identifying venoms in blood samples (from spiked and venom-challenged blood samples) of BALB/c mice, providing compelling evidence of the strategy's usefulness. This assay could help physicians diagnose and manage victims of snakebites through the evaluation of clinical samples.

Evaluation of SARS-CoV-2-Specific IgY Antibodies: Production, Reactivity, and Neutralizing Capability against Virus Variants.

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319-519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD-lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD-lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays.

Real-world evidence of a novel tetravalent immunoglobulin Y effectiveness and safety in patients with the refractory Helicobacter pylori infection.

BACKGROUND: Refractory Helicobacter pylori (H. pylori) infection inevitably increase the difficulty of drug selection. Here, we described our experience with the use of a novel tetravalent IgY against H. pylori for the treatment of patients with refractory H. pylori infection. METHODS: Patients were randomly assigned to receive the standard quadruple therapy (amoxicillin, clarithromycin, omeprazole and bismuth potassium citrate ) for 2 weeks or 250 mg of avian polyclonal IgY orally twice a day for 4 weeks. The binding efficacy of IgY to H. pylori antigens was detected by western blotting13. C-urea breath test was performed to evaluate the eradication therap's efficacy. The side effects of IgY were evaluated via various routine tests. The questionnaire was used to gather clinical symptoms and adverse reactions. RESULTS: Western blot analysis showed that tetravalent IgY simultaneously bind to VacA, HpaA, CagA and UreB of H. pylori. Tetravalent IgY had an eradication rate of 50.74% in patients with refractory H. pylori and an inhibition rate of 50.04% against DOB (delta over baseline) of 13C-urea. The symptom relief rate was 61.76% in thirty-four patients with clinical symptoms, and no adverse reactions were observed during tetravalent IgY treatment period. CONCLUSIONS: Polyclonal avian tetravalent IgY reduced H. pylori infection, and showed good efficacy and safety in the treatment of refractory H. pylori infection patients, which represented an effective therapeutic option of choice for patients with refractory H. pylori infection.

Rapid development and mass production of SARS-CoV-2 neutralizing chicken egg yolk antibodies with protective efficacy in hamsters.

Despite the record speed of developing vaccines and therapeutics against the SARS-CoV-2 virus, it is not a given that such success can be secured in future pandemics. In addition, COVID-19 vaccination and application of therapeutics remain low in developing countries. Rapid and low cost mass production of antiviral IgY antibodies could be an attractive alternative or complementary option for vaccine and therapeutic development. In this article, we rapidly produced SARS-CoV-2 antigens, immunized hens and purified IgY antibodies in 2 months after the SARS-CoV-2 gene sequence became public. We further demonstrated that the IgY antibodies competitively block RBD binding to ACE2, neutralize authentic SARS-CoV-2 virus and effectively protect hamsters from SARS-CoV-2 challenge by preventing weight loss and lung pathology, representing the first comprehensive study with IgY antibodies. The process of mass production can be easily implemented in most developing countries and hence could become a new vital option in our toolbox for combating viral pandemics. This study could stimulate further studies, optimization and potential applications of IgY antibodies as therapeutics and prophylactics for human and animals.

A study of the immunomodulatory effects of coconut oil extract in broilers experimentally infected with velogenic Newcastle disease virus.

OBJECTIVE: This study aims to evaluate the immunomodulatory effects of coconut oil extract (COE) in broilers experimentally infected with velogenic Newcastle disease virus (vNDV). METHODS: A total of 150 broiler birds (day-old) were equally divided into five study groups i.e., negative control, positive control, COE-1, COE-2, and COE-3. On day 10, broilers of groups COE-1, COE-2, and COE-3 were supplemented with 1, 2, and 3 mL of COE respectively per liter of drinking water for 15 days. On day 13, 0.1 mL/bird (10-5.25 ELD50) of vNDV was inoculated in broilers of positive control, COE-1, COE-2, and COE-3 groups intramuscularly. During this study, growth performance, morbidity, and mortality rates of each study group were recorded. The antibody titer against NDV was determined on days 7, 14, 21, 28, and 35. The levels of immunoglobulins (IgY and IgM) were also determined on the 7th, 14th, and 21st days post-sheep red blood cells (SRBCs) inoculation. On day 33, avian tuberculin was injected between the 1st and 2nd toes of the left side (intradermally) to measure lymphoproliferative responses. On day 35, the phagocytic activity in the blood was assessed through a carbon clearance assay by injecting carbon black ink into the right-wing vein. The visceral organs having gross lesions were also collected for histopathology. RESULTS: The COE significantly improved the growth performance, and lowered the morbidity and mortality rates of broilers. There was a significant rise in antibody titers against NDV and levels of IgY and IgM antibodies against SRBC in COE-supplemented broilers. The lymphoproliferative response and phagocytic activity were also enhanced. Among COEsupplemented groups, the broilers of the COE-3 group showed a significant increase in growth performance and boosted immune defense. CONCLUSION: Coconut oil extract has the potential to boost the growth performance and immune status of broilers. It can be used effectively as a feed additive and alternative to antibiotics to prevent the spread of infectious poultry pathogens.

Use of Immunoglobulin Y Antibodies: Biosensor-based Diagnostic Systems and Prophylactic and Therapeutic Drug Delivery Systems for Viral Respiratory Diseases.

Respiratory viruses have caused many pandemics from past to present and are among the top global public health problems due to their rate of spread. The recently experienced COVID-19 pandemic has led to an understanding of the importance of rapid diagnostic tests to prevent epidemics and the difficulties of developing new vaccines. On the other hand, the emergence of resistance to existing antiviral drugs during the treatment process poses a major problem for society and global health systems. Therefore, there is a need for new approaches for the diagnosis, prophylaxis, and treatment of existing or new types of respiratory viruses. Immunoglobulin Y antibodies (IgYs) obtained from the yolk of poultry eggs have significant advantages, such as high production volumes, low production costs, and high selectivity, which enable the development of innovative and strategic products. Especially in diagnosing respiratory viruses, antibody-based biosensors in which these antibodies are integrated have the potential to provide superiority in making rapid and accurate diagnosis as a practical diagnostic tool. This review article aims to provide information on using IgY antibodies in diagnostic, prophylactic, and therapeutic applications for respiratory viruses and to provide a perspective for future innovative applications.

Characterization of anti-soybean agglutinin (SBA) IgY antibodies: a new strategy for neutralization of the detrimental biological activity of SBA.

Anti-soybean agglutinin (SBA) IgY was produced, and its potential to neutralize the haemagglutinating activity of SBA in vitro was tested. Thirty-five-week-old hens [treatment (n = 5) and control (n = 5)] were immunized with SBA or injected with saline 4 times every 15 days. Eggs were collected after the last immunization, and IgY was extracted using the polyethylene glycol (PEG) method. Serum anti-SBA IgY titres in immunized hens increased after the first immunization and reached a plateau between days 45 and 60. In contrast, specific IgY titres in the control group remained at basal levels throughout the evaluation. Average IgY titres were significantly higher in the treatment group on days 15, 30, 45, and 60. Total IgY content in the egg yolk extract was 38.7 ± 1.6 and 37.7 ± 1.5 mg/ml for the treatment and control groups, respectively. The specific anti-SBA IgY titer detected in the egg yolk extract was significantly higher (p < 0.001) for hens in the treatment group compared to the control group, with OD450nm values of 0.98 ± 0.05 and 0.058 ± 0.02, respectively. The specificity of anti-SBA IgY was confirmed by the Western blotting, and the inhibition of SBA-induced haemagglutination in vitro was compared with D-galactose, a known molecule that binds to SBA and blocks its binding to erythrocytes. The inhibition of SBA-induced haemagglutination by the anti-SBA IgY reached 512 units of haemagglutination inhibition (UHI), compared to 8 or 256 UHI, respectively, when IgY from control chickens or D-galactose was used. Thus, anti-SBA IgY antibodies were efficiently produced in large quantities and effectively inhibited SBA-induced haemagglutination in vitro.

Protective effect of chicken yolk antibody Y against Campylobacter jejuni induced diarrhea in cats.

Campylobacter jejuni (C. jejuni) is a common pathogen that often causes diarrhea, loss of appetite, and even enteritis in domestic cats, affecting their growth and development, especially in kittens under 6 months of age. Oral passive immunization with chicken yolk antibody Y has been proved effective for the treatment of gastrointestinal pathogen infections due to its high specificity. In this study, C. jejuni was isolated from diarrheal cat feces, and the specific egg yolk antibody Y against C. jejuni was demonstrated to effectively inhibit its proliferation in vitro experiments. To evaluate the effect of anti-C. jejuni IgY, the mouse C. jejuni infection model was established and it was found that IgY could alleviate C. jejuni-induced clinical symptoms. Consistent with these results, the reduction of pro-inflammatory factors and intestinal colonization by C. jejuni in the IgY-treated groups, especially in the high dose group. We then evaluated the protective effect of IgY on young Ragdoll cats infected with C. jejuni. This specific antibody reduced the rate of feline diarrhea, protected the growth of young cats, inhibited systemic inflammatory hyperactivation, and increased fecal short-chain fatty acid concentrations. Notably, IgY may have a protective role by changing intestinal amino acid metabolism and affecting C. jejuni chemotaxis. Collectively, specific IgY is a promising therapeutic strategy for C. jejuni-induced cat diarrhea.

Preparation of Bispecific IgY-scFvs Inhibition Adherences of Enterotoxigenic Escherichia coli (K88 and F18) to Porcine IPEC-J2 Cell.

Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 µg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

FcRY is a key molecule controlling maternal blood IgY transfer to yolks during egg development in avian species.

Maternal immunoglobulin transfer plays a key role in conferring passive immunity to neonates. Maternal blood immunoglobulin Y (IgY) in avian species is transported to newly-hatched chicks in two steps: 1) IgY is transported from the maternal circulation to the yolk of maturing oocytes, 2) the IgY deposited in yolk is transported to the circulation of the embryo via the yolk sac membrane. An IgY-Fc receptor, FcRY, is involved in the second step, but the mechanism of the first step is still unclear. We determined whether FcRY was also the basis for maternal blood IgY transfer to the yolk in the first step during egg development. Immunohistochemistry revealed that FcRY was expressed in the capillary endothelial cells in the internal theca layer of the ovarian follicle. Substitution of the amino acid residue in Fc region of IgY substantially changed the transport efficiency of IgY into egg yolks when intravenously-injected into laying quail; the G365A mutant had a high transport efficiency, but the Y363A mutant lacked transport ability. Binding analyses of IgY mutants to FcRY indicated that the mutant with a high transport efficiency (G365A) had a strong binding activity to FcRY; the mutants with a low transport efficiency (G365D, N408A) had a weak binding activity to FcRY. One exception, the Y363A mutant had a remarkably strong binding affinity to FcRY, with a small dissociation rate. The injection of neutralizing FcRY antibodies in laying quail markedly reduced IgY uptake into egg yolks. The neutralization also showed that FcRY was engaged in prolongation of half-life of IgY in the blood; FcRY is therefore a multifunctional receptor that controls avian immunity. The pattern of the transport of the IgY mutants from the maternal blood to the egg yolk was found to be identical to that from the fertilized egg yolk to the newly-hatched chick blood circulation, via the yolk sac membrane. FcRY is therefore a critical IgY receptor that regulates the IgY uptake from the maternal blood circulation into the yolk of avian species, further indicating that the two steps of maternal-newly-hatched IgY transfer are controlled by a single receptor.

Ultrasensitive colorimetric detection of Staphylococcus aureus using wheat germ agglutinin and IgY as a dual-recognition strategy.

A novel colorimetric platform was designed for the determination of S. aureus by utilizing a dual-recognition strategy, where wheat germ agglutinin (WGA)-functionalized magnetic beads were served as separation elements to capture and enrich S. aureus efficiently from the matrix. Horseradish peroxidase (HRP) labeled chicken anti-protein A IgY (HRP-IgY) was used to label the captured S. aureus. A chicken IgY was introduced as a signal tracer to bind with staphylococcal protein A (SPA) on the surface of S. aureus, which can circumvent the interference from protein G-producing Streptococcus. Subsequently, the colorimetric signal was achieved by an HRP-catalyzed reaction, which was amplified by HRP-IgY bound by approximately 80,000 SPA molecules on one S. aureus. The entire detection process could be accomplished within 90 min. Under optimal conditions, the linear response of different S. aureus concentrations ranged from 7.8 × 102 to 2.0 × 105 CFU/mL and the limit of detection reached down to 3.9 × 102 CFU/mL. Some common non-target bacteria yielded negative results, indicating the excellent specificity of the method. The developed strategy was successfully applied to the determination of S. aureus in various types of samples with satisfactory recoveries. Therefore, the novel dual-recognition strategy possessed the advantages of high sensitivity, specificity, and low cost and exhibited considerable potential as a promising tool to defend public health.

H9N2 Avian Influenza Virus Downregulates FcRY Expression in Chicken Macrophage Cell Line HD11 by Activating the JNK MAPK Pathway.

The H9N2 avian influenza virus causes reduced production performance and immunosuppression in chickens. The chicken yolk sac immunoglobulins (IgY) receptor (FcRY) transports from the yolk into the embryo, providing offspring with passive immunity to infection against common poultry pathogens. FcRY is expressed in many tissues/organs of the chicken; however, there are no reports investigating FcRY expression in chicken macrophage cells, and how H9N2-infected HD11 cells (a chicken macrophage-like cell line) regulate FcRY expression remains uninvestigated. This study used the H9N2 virus as a model pathogen to explore the regulation of FcRY expression in avian macrophages. FcRY was highly expressed in HD11 cells, as shown by reverse transcription polymerase chain reactions, and indirect immunofluorescence indicated that FcRY was widely expressed in HD11 cells. HD11 cells infected with live H9N2 virus exhibited downregulated FcRY expression. Transfection of eukaryotic expression plasmids encoding each viral protein of H9N2 into HD11 cells revealed that nonstructural protein (NS1) and matrix protein (M1) downregulated FcRY expression. In addition, the use of a c-jun N-terminal kinase (JNK) activator inhibited the expression of FcRY, while a JNK inhibitor antagonized the downregulation of FcRY expression by live H9N2 virus, NS1 and M1 proteins. Finally, a dual luciferase reporter system showed that both the M1 protein and the transcription factor c-jun inhibited FcRY expression at the transcriptional level. Taken together, the transcription factor c-jun was a negative regulator of FcRY, while the live H9N2 virus, NS1, and M1 proteins downregulated the FcRY expression through activating the JNK signaling pathway. This provides an experimental basis for a novel mechanism of immunosuppression in the H9N2 avian influenza virus.

Evaluation of anti-Fel d 1 IgY ingredient for pet food on growth performance in kittens.

Introduction: The domestic cat (Felis catus) is one of the most common pets. Worldwide, approximately one in five adults are sensitive to cat allergens. The major cat allergen is the secretoglobulin Fel d 1, which is primarily produced in the salivary and sebaceous glands. Chickens produce IgY antibodies, which are similar in structure to mammalian IgG. When chickens are exposed to Fel d 1, anti-Fel d 1-specific IgY (AFD1) is produced and is naturally concentrated in egg yolk. The aim of this study was to evaluate the tolerability, effects on growth and food consumption, and potential adverse effects of a chicken egg product ingredient containing AFD1 in kittens. Methods: This was a blinded, controlled study. Twenty-seven (27) eight-week old kittens were randomly assigned to three feeding groups containing 0 ppm AFD1 (Group 0), 8 ppm AFD1 (Group 1), and 16 ppm AFD1 (Group 2) for 84 days. Veterinary exams and bloodwork were performed on Day 42 and Day 84, and body weight and body condition score (BCS) were monitored weekly. Results: Throughout the study, there were no signs of nutritional deficiency or adverse clinical events in any of the subjects. Administration of a chicken egg product ingredient containing AFD1 in the diet (whether in coating or combination of coating and top dress) had no significant effect on body weight nor food consumption, and all subjects maintained a healthy Body Condition Score (BCS) throughout the study. Moreover, there were no biologically significant differences in the mean clinical chemistry and hematology parameters. Discussion: This study demonstrated that a diet formulated to contain up to 16 ppm AFD1, included in the coating and the top-dress of dry kitten food, was well tolerated, promoted adequate growth, and exhibited no adverse effects.

Production of egg yolk antibody (IgY) against a chimeric protein containing IpaD, StxB, and TolC antigens from Shigella: An investigation of its prophylactic effects against Shiga toxin (Stx) and Shigella dysenteriae in vitro and in vivo.

Shigella is a major problem in developing countries. Immunoglobulin Y (IgY) can be used for prophylaxis and neutralize bacteria. The aim of this study was to produce IgY against the chimeric protein containing IpaD, StxB, and TolC antigens from Shigella, investigate its prophylactic and neutralizing effects against Stx and Shigella dysenteriae. The nucleotide sequence corresponding to the chimeric protein was cloned into pET28a plasmid and expressed in E. coli BL21 (DE3). Protein expression was confirmed by SDS-PAGE and the recombinant protein was purified by Ni-NTA affinity chromatography. The 150 µg of chimeric protein was mixed with Freund's adjutant and injected into laying hens (Leghorn). IgY was purified using PEG6000 precipitation. Antibody titer in the serum and egg yolk was evaluated by ELISA. IgY challenge against 1,10 and 50 LD50 of Stx and S. dysenteriae was investigated. A 60.6 kDa recombinant protein was confirmed by SDS-PAGE. ELISA showed that the antibody titer was significantly increased. MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] showed that at 16 µmol/L, IgY protected HeLa cells against Stx. Treatment of mice with 1000 and 1500 µg IgY leads to complete survival of the mice against 1LD50 toxin and 4000 µg of IgY led to complete survival against 1LD50, also 70% and 30% survival against 10 and 50 LD50S. dysenteriae. This study showed that IgY produced against Stx and Shigella virulence factors could cause high protective effects against bacteria and toxins.