Yolk Immunoglobulin Y Research Articles

Egg Yolk Antibody for Passive Immunization: Status, Challenges, and Prospects.

The immunoglobulin Y (IgY) derived from hyperimmune egg yolk is a promising passive immune agent to combat microbial infections in humans and livestock. Numerous studies have been performed to develop specific egg yolk IgY for pathogen control, but with limited success. To date, the efficacy of commercial IgY products, which are all delivered through an oral route, has not been approved or endorsed by any regulatory authorities. Several challenging issues of the IgY-based passive immunization, which were not fully recognized and holistically discussed in previous publications, have impeded the development of effective egg yolk IgY products for humans and animals. This review summarizes major challenges of this technology, including in vivo stability, purification, heterologous immunogenicity, and repertoire diversity of egg yolk IgY. To tackle these challenges, potential solutions, such as encapsulation technologies to stabilize IgY, are discussed. Exploration of this technology to combat the COVID-19 pandemic is also updated in this review.

Impact of Dietary Egg Yolk IgY Powder on Behavior, Meat Quality, Physiology, and Intestinal Escherichia coli Colonization of Broiler Chicks.

The current study investigated the impact of different concentrations of purified egg yolk immunoglobulin Y (IgY) supplemental food on the growth performance, behaviors, cecal contents of Escherichia coli, and the meat quality of broiler chicks. Four dietary groups were given to 180 female Ross broiler chicks at random (n = 45 for each). The control group was fed a standard diet only, whereas the other three experimental groups were fed the same basic diet supplemented with 1,500, 3,000, and 4,000 μg/ml IgY for a duration of 42 days. Significant greater behavioral activities, including, feeding, drinking, and dust bathing (p < 0.05), in the birds fed 4,000 μg/ml of IgY compared to the control group were observed. Greater weight gains of the crop, proventriculus, gizzard, and intestine (p < 0.05) were observed for broiler chicks fed 4,000 μg/ml of IgY when compared to the control group. After 3 weeks of feeding, the groups fed 3,000 and 4,000 μg/ml IgY had significant lower E. coli counts in the muscle and cecal contents (p < 0.05) when compared to the control group. Moreover, dietary supplementation with 4,000 μg/ml IgY in the third week and 3,000 μg/ml IgY in the sixth week resulted in greater weight gain (p < 0.01) when compared to the control group. Also, at week 3, chicks fed 4,000 μg/ml of IgY had a lower feed conversion ratio (FCR) when compared to the control group (p < 0.05). At week 6, chicks fed 3,000 μg/ml of IgY had lower FCR than the control (p < 0.05). The circulating heterophile/lymphocyte ratio was simply altered in birds fed variable IgY concentrations (1,500, 3,000, and 4,000 μg/ml), with no significant differences compared to the control group due to the individual resistance of each bird to physiological stress. The addition of 4,000 μg/ml IgY to the diet enhanced the nutritive value of meat, including protein, fat, and ash content (p < 0.05). Our study concluded that dietary supplementation of 3,000 and/or 4,000 μg/ml IgY improved the growth rates, behavioral activities, intestinal health indices, and meat quality of broiler chicks.

Passive Immunization of Chickens with Anti-Enterobactin Egg Yolk Powder for Campylobacter Control.

Enterobactin (Ent) is a highly conserved and important siderophore for the growth of many Gram-negative bacterial pathogens. Therefore, targeting Ent for developing innovative intervention strategies has attracted substantial research interest in recent years. Recently, we developed a novel Ent conjugate vaccine that has been demonstrated to be effective for controlling Gram-negative pathogens using both in vitro and in vivosystems. In particular, active immunization of chickens with the Ent conjugate vaccine elicited strong immune responses and significantly reduced intestinal colonization of Campylobacter jejuni, the leading foodborne bacterial pathogen. Given that hyperimmune egg yolk immunoglobulin Y (IgY) has been increasingly recognized as a promising and practical non-antibiotic approach for passive immune protection against pathogens in livestock, in this study, we assessed the efficacy of oral administration of broiler chickens with the anti-Ent hyperimmune egg yolk powder to control C. jejuni colonization in the intestine. However, supplementation of feed with 2% (w/w) of anti-Ent egg yolk powder failed to reduce C. jejuni colonization when compared to the control group. Consistent with this finding, the ELISA titers of the specific IgY in cecum, ileum, duodenum, gizzard, and serum contents were similar between the two groups throughout the trial. Chicken intestinal microbiota also did not change in response to the egg yolk powder treatment. Subsequently, to examine ex vivo stability of the egg yolk IgY, the chicken gizzard and duodenum contents from two independent sources were spiked with the egg yolk antibodies, incubated at 42 °C for different lengths of time, and subjected to ELISA analysis. The specific IgY titers were dramatically decreased in gizzard contents (up to 2048-fold) but were not changed in duodenum contents. Collectively, oral administration of broiler chickens with the anti-Ent egg yolk powder failed to confer protection against intestinal colonization of C. jejuni, which was due to instability of the IgY in gizzard contents as demonstrated by both in vivo and ex vivo evidence.

Egg Yolk IgY: A Novel Trend of Feed Additives to Limit Drugs and to Improve Poultry Meat Quality.

Drugs that are commonly used in poultry farms can potentially cause a detrimental effect on meat consumers as a result of chemical residues. Therefore, seeking a natural alternative is crucial for the health of the consumers. The egg yolk immunoglobulin Y (IgY) is a promising natural replacement for antibiotics in the broilers' diet. There is a scarce focus on the influence of probiotics and IgY on the quality and the nutritive values of broiler meat and whether it can efficiently displace the anti-microbial power of antibiotics. Herein we used 40 Ross chicks (1.2 ± 0.43 days old) and separated them into four groups with variant feed additives (basal diet “control,” probiotic, IgY, and probiotic + IgY). Our findings showed that the combination of probiotic and IgY supplementation enhanced the carcass quality traits and decreased the pH values that could retard spoilage due to bacteria and improve shelf life and meat quality. The same group also achieved a significant reduction in thiobarbituric acid value, indicating an improvement of meat quality. Moreover, color, shear force, water holding capacity, and cooking loss were most acceptable in broiler meat supplemented with IgY, which confirmed the highest carcass quality. Notably, the weight gain in the combination group has been greatly increased. Also, the protein percentage was the highest (22.26 ± 0.29, P < 0.001) in this combined supplementation group, which revealed the highest nutritive values. Staphylococcus aureus and Escherichia coli could not be detected in the meat of the probiotics group and/or in the combined treatment group. Interestingly, the IgY group showed an evidence of the killing power (log colony-forming units per milliliter) of S. aureus and Listeria monocytogenes at 1,500 μg/ml. Our findings, in vitro as well as in vivo, revealed that the combination group had antimicrobial bioactivity and enhanced the chickens' immunity. Therefore, IgY, a novel trend of feed additives, can be used to limit drugs. Additionally, the mortality percentage recorded was zero in all groups that received feed supplementation, while the combination group reached the best financial advantages. We concluded that feeding IgY powder with probiotic is a frontier to improve the productivity, immunity, and meat quality of broilers.

The Impact of Probiotics and Egg Yolk IgY on Behavior and Blood Parameters in a Broiler Immune Stress Model.

Feed additives are used frequently in variable combinations to maximize broiler productivity and consumer safety. Therefore, we evaluated the efficiency of feed additives used in four different diets: a basal diet, a probiotic (PRO-PAC®) supplement diet, an egg yolk purified immunoglobulin Y (IgY) supplemented diet, and a combination of IgY and PRO-PAC® supplement (n = 15 for each group). We assessed the improvement of behavioral and hematological parameters of Ross broilers before and after an immune stress challenge using lipopolysaccharide (LPS). Behavioral as well as physiological parameters were analyzed. The standing frequency was the highest (P < 0.05) in broilers supplemented with a combination of probiotics and IgY. Likewise, latency approach score to a novel object improved (P < 0.01) in the combination group at week-3. After intraperitoneal injection of LPS, this combination group achieved the best gait score at week-3, followed by week-5, compared to birds fed the basal diet. The heterophil/lymphocyte (H/L) ratio, heterophil differential count, and eosinophil differential count in the basal diet group that was challenged with LPS were significantly increased (P < 0.01, P < 0.001, P < 0.05, respectively) compared to the combination groups. Therefore, we concluded that the combination of IgY and probiotics can significantly improve the behavior and the underlying physiological parameters of Ross broilers. Consequently, this combination can improve the broilers′ health, welfare and produce a safe meat free from harmful chemical residues.

Evaluation of a receptor gene responsible for maternal blood IgY transfer into egg yolks using bursectomized IgY-depleted chickens

In avian species, maternal immunoglobulin Y (IgY) is transferred from the blood to the yolks of maturing oocytes; however, the mechanism underlying this transfer is unknown. To gain insight into the mechanisms of maternal IgY transfer into egg yolks, IgY-depleted chickens were generated by removing the bursa of Fabricius (bursectomy) during egg incubation, and their egg production and IgY transport ability into egg yolks were determined. After hatching, blood IgY concentrations of the bursectomized chickens decreased gradually until sexual maturity, whereas those of IgA remained low from an early stage of growth (from at least 2 wk of age). Chickens identified as depleted in IgY through screening of blood IgY and IgA concentrations were raised to sexual maturity. At 20 wk of age, both blood and egg yolk IgY concentrations in the IgY-depleted group were 600-fold lower than those of the control group, whereas egg production did not differ between the groups. Intravenously injected, digoxigenin-labeled IgY uptake into the egg yolk was approximately 2-fold higher in the IgY-depleted chickens than in the controls, suggesting that IgY depletion may enhance IgY uptake in maturing oocytes. DNA microarray analysis of the germinal disc, including the oocyte nucleus, revealed that the expression levels of 73 genes were upregulated more than 1.5-fold in the IgY-depleted group, although we could not identify a convincing candidate gene for the IgY receptor. In conclusion, we successfully raised IgY-depleted chickens presenting a marked reduction in egg yolk IgY. The enhanced uptake of injected IgY into the egg yolks of the IgY-depleted chickens supports the existence of a selective IgY transport mechanism in maturing oocytes and ovarian follicles in avian species.

IgY antibody and human neurocysticercosis: a novel approach on immunodiagnosis using Taenia crassiceps hydrophobic antigens.

Human neurocysticercosis (NCC) is a worldwide neglected disease caused by Taenia solium metacestode and responsible for various complications and neurological disorders. This study aimed to evaluate the use of specific immunoglobulin Y (IgY) produced by laying hens immunized with a hydrophobic fraction of Taenia crassiceps metacestodes (hFTc) in NCC diagnosis. Egg yolk IgY antibodies were fractionated, purified and characterized. Enzyme-linked immunosorbent assay (ELISA) was carried out to evaluate the production kinetics and avidity maturation of anti-hFTc IgY antibodies throughout the IgY obtention process. Antigen recognition tests were carried out by Western blotting and immunofluorescence antibody test using purified and specific anti-hFTc IgY antibodies for detection of parasitic antigens of T. crassiceps and T. solium metacestodes. Sandwich ELISA was performed to detect circulating immune complexes formed by IgG and parasitic antigens in human sera. The results showed high diagnostic values (93.2% sensitivity and 94.3% specificity) for immune complexes detection in human sera with confirmed NCC. In conclusion, specific IgY antibodies produced from immunized hens with hFTc antigens were efficient to detect T. solium immune complexes in human sera, being an innovative and potential tool for NCC immunodiagnosis.

Efficient Production of Human Norovirus-Specific IgY in Egg Yolks by Vaccination of Hens with a Recombinant Vesicular Stomatitis Virus Expressing VP1 Protein.

Human norovirus (HuNoV) is responsible for more than 95% of outbreaks of acute nonbacterial gastroenteritis worldwide. Despite major efforts, there are no vaccines or effective therapeutic interventions against this virus. Chicken immunoglobulin Y (IgY)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases. Here, we developed a highly efficient bioreactor to generate high titers of HuNoV-specific IgY in chicken yolks using a recombinant vesicular stomatitis virus expressing HuNoV capsid protein (rVSV-VP1) as an antigen. We first demonstrated that HuNoV VP1 protein was highly expressed in chicken cells infected by rVSV-VP1. Subsequently, we found that White Leghorn hens immunized intramuscularly with rVSV-VP1 triggered a high level of HuNoV-specific yolk IgY antibodies. The purified yolk IgY was efficiently recognized by HuNoV virus-like particles (VLPs). Importantly, HuNoV-specific IgY efficiently blocked the binding of HuNoV VLPs to all three types (A, B, and O) of histo-blood group antigens (HBGAs), the attachment factors for HuNoV. In addition, the receptor blocking activity of IgY remained stable at temperature below 70 °C and at pH ranging from 4 to 9. Thus, immunization of hens with VSV-VP1 could be a cost-effective and practical strategy for large-scale production of anti-HuNoV IgY antibodies for potential use as prophylactic and therapeutic treatment against HuNoV infection.

An ELISA for quantifying quail IgY and characterizing maternal IgY transfer to egg yolk in several quail strains

In avian species, maternal blood immunoglobulin Y (IgY) is transferred to the egg yolks of maturing oocytes, but the mechanism underlying this transfer is unknown. To gain insight into the mechanism of maternal IgY transfer in quail, we established an enzyme-linked immunosorbent assay (ELISA) for the quantitation of quail IgY. We characterized strain differences in blood and egg yolk IgY concentrations and exogenously injected IgY-Fc uptakes into egg yolks. A specific rabbit polyclonal antibody to quail IgY was raised for the ELISA. Blood and egg yolk IgY concentrations were determined in six quail strains (one inbred strain, L; four closed population strains, AWE, DB, PS, WE; one commercial strain, Commercial). The birds were also injected with digoxigenin-labeled quail IgY-Fc, and its uptakes into laid eggs were compared. The strain difference in blood and egg yolk IgY concentrations was at most 2.5-fold, between PS and AWE. The rank order of IgY concentrations was AWE, Commercial, DB, L≥WE≥PS. A significant positive correlation (|R|=0.786) between individual blood IgY and egg yolk IgY and the concentrated egg yolk IgY (1.5–2-fold) against blood IgY was observed. Interestingly, there was a significant inverse correlation (|R|=0.452) between injected IgY-Fc uptakes and the blood IgY concentration, implying competition of the injected IgY-Fc and blood IgY in the process of IgY uptake into egg yolks. In conclusion, we successfully determined blood and egg yolk IgY concentrations in various quail strains by a quail IgY-specific ELISA. The concentrated egg yolk IgY against the blood IgY and the inverse relationship of exogenous IgY-Fc uptake against the blood IgY supports the existence of a selective IgY transport mechanism in avian maturing oocytes.

Comparative study on immunoglobulin Y transfer from breeding hens to egg yolk and progeny chicks in different breeds of poultry.

Aim:This study was undertaken to compare the immunoglobulin Y (IgY) level and its efficacy in laying hens of four different breeds of poultry (viz., Vanraja, Gramapriya, BlackRock, and KalingaBrown) and its relative transfer in egg yolk and chick.Materials and Methods:This study was conducted in 48 apparently healthy laying hens vaccinated with Salmonella inactivated polyvalent vaccine, eggs and progeny chicks; 12 each from four different breeds of poultry, viz., Vanraja, Gramapriya, BlackRock, and KalingaBrown. The methodology included measurement of egg and yolk weight, total protein and IgY in egg yolk, total serum protein and IgY in breeding hens, and progeny chicks and extent of IgY transfer from hens to yolk then to chicks. Further, Salmonella-specific antibodies in breeding hens, egg yolk and progeny chicks were assessed using O and H antigen by tube agglutination test.Results:The egg weight differed nonsignificantly (p>0.05) among breeds, however, breed wise significant variation (p<0.01) was reported in yolk weight. The weight of egg yolk significantly affects the total protein and IgY concentration although these levels per unit of volume did not differ. Total protein was significantly higher (p<0.01) in KalingaBrown and Gramapriya as compared to Vanraja and BlackRock. Non-significant (p>0.05) difference among breed was found in total protein of egg yolk and chick. The IgY concentration in hens, egg yolk and chick was found to be in the range of 5.35±0.63-5.83±0.65, 2.3±0.1-2.6±0.2, and 1.3±0.11-1.7±0.16 mg/ml, respectively which is uniform and independent of total protein concentration at all the three levels. Significant breed variations were not observed in maternal IgY transfer from breeding hens to chicks and were 25.62±1.42-36.06±4.34% of total IgY in parent flock. Moderate to higher rate of seroprevalence with peak titers of 1:640 against Salmonella-specific antibodies was observed in only 41.6% of breeding hens.Conclusion:No significant difference in the rate of transfer of IgY was observed in four breeds studied (viz., Vanraja, Gramapriya, BlackRock, and KalingaBrown) and moderate seropositivity was detected for Salmonella-specific antibodies in progeny chicks.

Evaluation of IgY capture ELISA for sensitive detection of Alpha hemolysin of Staphylococcus aureus without staphylococcal protein A interference

Staphylococcal protein A (Spa) secreted by all Staphylococcus aureus strains is the major hindrance in development of specific immunoassays for detecting S. aureus antigens, because of its characteristic feature of binding to Fc region of most mammalian immunoglobulins and also to Fab region of certain classes of mammalian immunoglobulins. Immunoglobulin Y (IgY) is the avian equivalent of mammalian IgG which does not have any affinity to Spa. In the present study we report that using chicken egg yolk IgY over mammalian IgG as capture antibody prevents both soluble and surface bound protein A from causing false positives quantified by chicken anti-protein A antibodies. This was demonstrated by development of sandwich ELISA for detection of alpha hemolysin toxin from culture supernatants of S. aureus strains with anti alpha hemolysin IgY as capture and rabbit anti alpha hemolysin IgG as revealing antibody. This indirect sandwich ELISA was evaluated onto a large number of S. aureus isolates recovered from clinical sources for alpha hemolysin secretion. Results of sandwich ELISA were compared with PCR and Western blot analysis. The immunoassay is highly specific and has high sensitivity of detecting less than 1ng/ml. This procedure is highly effective in eliminating Spa interference and can be extended to detection of other important superantigen toxins of S. aureus.

Determination of Immunoglobulin Y Concentration in Yolk Extract Prepared by Water Dilution Method: Comparisons among Three Strains of Chickens

In avian species, immunoglobulin Y (IgY) is found abundantly in egg yolk as well as in blood. Although the concentration of IgY in yolk has been previously reported by many investigators, the reported IgY concentrations have varied from 1 to 25mg/g yolk. In the present study, we determined yolk IgY concentration in three strains of chickens, taking into consideration the IgY recovery rate in yolk extract; in addition, we estimated the concentration factor of yolk IgY against plasma IgY. Egg yolk and plasma samples were collected from two commercial layers, Dekalb and Nagoya, and one inbred strain, PNP/DO. Extraction of yolk IgY was followed by the water dilution method reported by Akita and Nakai (Journal of Food Science 57, 629-634, 1992) with minor modification, and IgY concentration was measured by ELISA. The recovery of IgY from the yolk was in the 50 to 60% range, and no significant difference was observed among the strains. PNP/DO showed the highest concentration of IgY expressed as mg/g yolk (12.2), and Dekalb and Nagoya had almost the similar concentration of IgY (Dekalb, 6.2; Nagoya, 5.7). In all strains, the concentration of plasma IgY was proportional to that of yolk IgY, and the concentration factor of yolk IgY against plasma IgY was approximately 0.8. On the other hand, when the yolk IgY concentration was expressed as mg/mL water in yolk, the concentration factor of yolk IgY against plasma IgY represented approximately 1.7 regardless of the strains. Taken together, our findings suggest that the concentration of IgY in the yolk should be determined by considering the recovery rate during the process of IgY extraction; in addition, it raised the possibility that blood IgY might be concentrated in egg yolks of chickens.

Influence of Aging and Estrogen Treatment on the IgY Concentration in the Egg Yolk of Chicken, Gallus domesticus.

In laying hens, immunoglobulin G (IgG) is transferred from maternal body to the egg yolk and termed as immunoglobulin Y (IgY). The goal of this study was to determine the effects of aging and estrogen treatment on the concentration of IgY in egg yolk. The concentration of yolk IgY and plasma IgG of young laying (approximately 170-day-old), middle-aged laying (approximately 250-day-old) and old laying (approximately 575-day-old) hens as well as old laying hens (approximately 575-day-old) treated with diethylstilbestrol (DES, an estrogenic compound) for 7 days were determined by ELISA. Immunoglobulin Y concentration was significantly greater in young laying hens than in middle-aged and old laying hens, in contrast, plasma IgG concentration was greater in old laying hens than in middle-aged and young laying hens (P<0.01). The concentration of yolk IgY and plasma IgG was significantly increased after DES treatment as compared with pretreatment (P<0.01). Significant differences were not observed in the concentration of yolk IgY and plasma IgG between pre- and post-oil treatment of hens (control birds). These results indicate that the concentration of IgY decreases with aging of hens unlike plasma IgG concentration, and the concentration of IgY is significantly affected by estrogen treatment.

Production and purification of Fab′ fragments from chicken egg yolk immunoglobulin Y (IgY)

Methods were described for the production of Fab and Fab′ fragments from chicken egg yolk IgY also referred to as IgG by papain and pepsin digestion respectively. Pepsin digestion was found to be suitable for the large scale preparation and purification of Fab′. Optimum yield of Fab′ was obtained after peptic digestion of IgY at pH 4.2 for 9 h at low NaCl concentration. This condition led to the complete digestion of pFc′ fragment leaving only the Fab' fragment. By combination of ultrafiltration and anion exchange, and conditions which allowed binding of the small amount of contaminants in the digest to the anion exchange column, pure Fab′ fragments were easily obtained in the eluent. The advantage of this approach is that a small column could be used to purify large amount of protein, therefore, improving the efficiency of purification. The Fab and Fab′ fragments appeared to be similar on the basis of their molecular weights as determined by SDS-PAGE, reaction of identity in immunodiffusion assay and similar antigen binding activities as shown by ELISA.

Protective properties of egg yolk IgY containing anti‐Edwardsiella tarda antibody against paracolo disease in the Japanese eel, Anguilla japonica Temminck &amp; Schlegel

Abstract. Immunized hens are known to contain a high level of immunoglobulin Y (IgY) in their egg yolk. In this study, the present authors obtained anti‐Edwardsiella tarda IgY (containing 20% specific IgY, agglutination titre, 1:128) from hens vaccinated by injection with formalin‐killed bacterin. The IgY was stable against eel digestive factors, and therefore, was orally administered with viable E. tarda to the Japanese eels and the efficacy of protection against E. tarda infection was evaluated. Orally administered IgY at a dose of 400mg fish‐1 cleared E. tarda inoculated simultaneously at 105–6 CFU fish‐1 from the intestine within 24h. Moreover, orally administered IgY at doses of 200 and 400mg fish‐1 inhibited the penetration of E. tarda inoculated simultaneously at 104–6 CFU fish‐1 into the liver and kidney via the damaged intestine. The fish orally administered with IgY showed reduced mortality. These results suggest that egg yolk containing anti‐E. tarda IgY is effective in preventing edwardsiellosis.