Immunoglobulin Y Research Articles

Production and purification of Clostridium perfringens type D epsilon toxin and IgY antitoxin

The aim of this study was to purify Clostridium perfringens type D epsilon toxin and produce and purify anti-epsilon chicken immunoglobulin Y (IgY). A single-step ion exchange chromatography resulted in a high-yield and high-purity toxin, while ion exchange chromatography followed by gel filtration resulted in the highest purity of the toxin, but at a lower yield. Purified and inactivated epsilon toxin were then administered in chickens via four inoculations and IgY was obtained at a high purity and yield, with an antibody titer of 50 IU/mL and high levels of avidity (73.2%). In summary, C. perfringens type D epsilon toxin and chicken anti-epsilon IgY were successfully produced and purified, and may be used for the diagnosis of enterotoxemia caused by the epsilon toxin, as well as in potency tests of existing and future vaccines against enterotoxemia.

Serological evidence of avian HEV antibodies in apparently healthy chickens in southwest Nigeria.

Avian hepatitis E virus (aHEV) is associated with hepatitis-splenomegaly syndrome, big liver and spleen disease and hepatic rupture haemorrhage syndrome. However, the knowledge about aHEV in commercial layer chickens in Nigeria is scarce. In this study, 460 serum samples obtained from 36 apparently healthy commercial layer chicken flocks in three states (Ogun, Osun and Oyo States) of southwestern Nigeria were analysed by enzyme linked immunosorbent assay for the presence of anti-aHEV immunoglobulin Y (IgY) antibodies. In total, the overall seroprevalence of anti-aHEV antibodies was 14.6%. The serological analysis revealed that 75% of the flocks examined were positive for anti-aHEV IgY antibodies from chickens of various ages in all three states. The percentage of the seropositive chickens in the three states varied from flock to flock ranging from 60% to 88.8% and seropositive chickens were detected at any age (24–52 weeks of age) without significant differences between the age groups. This is the first report assessing the presence of aHEV antibodies in chickens from Nigeria. The detection of anti-aHEV antibodies in commercial layer chickens in this study emphasizes the importance of serosurveillance in disease monitoring due to the economic threat posed by aHEV as a result of decreased egg production and increased mortality in affected commercial layer chicken farms. However, further studies are essential to reveal the clinical implications and to assess the real burden of aHEV in Nigeria.

Molecular stability of the rabbit and chicken egg yolk immunoglobulins.

Antibodies play a vital role in a variety of applications from diagnostics, imaging, and therapeutics. The stability of antibodies is one of the most important key attributes for its prolonged activity and functionality. Here, we demonstrate a detailed comparative study of the molecular stability of the rabbit immunoglobulin G (IgG) and chicken egg yolk immunoglobulin Y (IgY) at different pH, temperatures, and time points. The molecular stability of IgG and IgY is compared on the basis of its binding activity and conformational changes. The optimum temperature and time were found to be 30 °C, and 37 °C, up to 8 h for both IgY and IgG antibodies. With increasing temperature and time, IgG antibodies were found to be significantly less stable in comparison to IgY antibodies. IgY is almost twenty-fold more stable than IgG at 60° C for up to 8 hours. The extra domain present in the heavy chain of IgY plays a significant role in providing more molecular stability as compared to IgG under the above-mentioned experimental conditions. The results show that, as compared with IgG, the IgY are more stable, are less expensive to make in high yield and exhibit minimal conformational changes and hence are cost effective for use in a diverse array of purposes.

Simple and efficient protocol for immunoglobulin Y purification from chicken egg yolk

Besides being a common food component broadly consumed worldwide, egg yolk immunoglobulin Y (IgY) has essential therapeutic potentials. In fact, in a time of ever-increasing risk of antibiotic resistance, it is crucial to find new ways to battle infection, and oral administration of preformed specific antibodies represents one of the most attractive approaches against infection. Infectious diseases of bacterial and viral origin in humans and animals can be controlled and passively cured by orally applied IgYs isolated from chicken egg yolks. Despite multiple obvious advantages of oral administration of IgY, harvesting IgY from egg yolk in a pure form is a challenging task.In this study, we developed a fast, simple, cost-effective, and efficient protocol for IgY isolation from chicken egg yolks. First, egg yolk was collected and diluted with 5 volumes of cold distilled water, homogenized, pH adjusted, and centrifuged. Next, the supernatant was collected, to which caprylic acid at concentration of 2% v/v was added, followed by pH adjustment to pH 5.0, centrifugation at 4°C, and collection of the resulting supernatant. This step was repeated twice, with adding 2% v/v of caprylic acid each time. The final supernatant was concentrated using ultrafiltration, and the IgY purity and activities were checked by SDS-PAGE, western blotting, and ELISA. The sequential (2, 2, 2%) addition of caprylic acid yielded IgY with a purity of 63.5, 90.6, and 95.8%, respectively, and reached 97.9% after ultrafiltration at pH 9.0. The IgY activity increased exponentially to reach 99% after the ultrafiltration step.The proposed caprylic-acid-based protocol of IgY purification from the yolk of chicken eggs seems to be simple, fast, direct, and very cheap. This indicates that this protocol has great potential for scale-up processing.

Evaluation of immunoglobulin-Y in place of tylosin phosphate in the diets fed to Holstein Steers and preliminary analysis of liver abscess duration on animal growth performance.

Despite the regular use of feed-grade macrolide-antibiotics, bovine liver abscesses persist, representing a financial burden to pre- and post-mortem sectors of the beef industry. An immunoglobulin-Y (IGY) additive developed to target Fusobacterium necrophorum and Trueperella pyogenes, was evaluated for the control of liver abscesses. Research is needed for the impact of liver abscess severity as well as abscess duration on steer performance and carcass characteristics. Holstein steers (n = 64; initial body weight (BW) = 372.5 ± 2.41 kg) consuming a finishing diet for 188 d were used in a completely randomized design where treatments included: TYL (tylosin phosphate 90 mg/d; n = 32) or IGY (2.5 g/d; n = 32) and steer was the experimental unit. Feed intake was recorded daily while BW and liver ultrasound outcome (normal or abnormal) was recorded every 28 d until slaughter to estimate duration of abscess presence (DUR). Continuous variables of animal growth performance and carcass characteristics were analyzed using the MIXED procedure of SAS. Categorical quality grade and liver data were analyzed using the GLIMMIX procedure of SAS. Treatment did not affect live or carcass-adjusted growth performance (P ≥ 0.131). However, steers fed TYL had greater (P = 0.042) empty body fat (EBF) % and a greater proportion of carcasses grading premium choice than steers fed IGY (P = 0.030). Treatment did not affect prevalence of abscessed livers, abscess severity or estimated abscess duration (P ≥ 0.213) but datasets with greater experimental units are needed to substantiate this outcome. Increasing abscess severity tended (P ≤ 0.10) to linearly reduce carcass-adjusted gain to feed (G:F), fat thickness, and EBF. Carcass dressing % was only affected by severe (A+ and A+AD) abscess scores (P = 0.010). Carcass-adjusted final BW, average daily gain, G:F, and hot carcass weight was decreased only when the estimated DUR was ≥140 d (P ≤ 0.05). Carcass dressing %, however, was linearly affected by estimated liver abscess DUR (P ≤ 0.005), regardless of abscess severity. Preliminary evidence suggests that measuring the duration of liver abscess affliction during the feeding period may also give insight to the degree of performance reduction.

82 Evaluation of feeding immunoglobulin-Y to Holstein feeder cattle for the control of liver abscesses & reduction of macrolide-resistant bacteria dissemination

Abstract Despite the regular use of macrolide-antibiotics in the diets of feedlot cattle, liver abscesses persist representing a financial burden to both pre- and post-mortem sectors of the beef industry. Alternative strategies, which simultaneously address liver infections and reduce the dissemination of antibiotic-resistant bacteria, are needed. An immunoglobulin-Y (IGY) product, developed to target Fusobacterium necrophorum and Trueperella pyogenes, was evaluated for the control of liver abscesses and its effect on macrolide-resistant Enterococcus growth from feces. Sixty-four Holstein steers (Initial BW= 384±13.5kg) consuming a finishing diet (90% concentrate) for 188 days (Final BW= 660±42 kg) were used in a completely randomized design where treatments included: TYL (Tylosin phosphate fed at 90 mg/hd/day; n = 32) or IGY (fed at 2.5g/hd/day; n = 32).Feed intake was recorded daily and BW was recorded every 28 days until slaughter. After 150 d on treatment fecal samples were randomly obtained from each treatment group four times (n = 40) and cultured on bile-esculine agar enriched with 8 μg/mL of erythromycin. Continuous variables of feedlot growth performance, carcass data and bacterial growth were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effects of steer or sampling day, respectively. Ordinal carcass data was analyzed using the FREQ procedure of SAS observing the Cochran-Mantel-Haenszel value. Treatment did not affect DMI (P = 0.21), ADG (P = 0.50) or G:F (P = 0.36). Treatment neither affected hot carcass weight (P = 0.37) nor longissimus muscle area (P = 0.25). However, TYL resulted in greater back fat thickness than IGY (0.32 vs. 0.23 in.; P < 0.02) and consequently greater numerical yield grade (P < 0.02). Quality grade was not affected by treatment (P = 0.65). No treatment difference was observed for liver abscesses prevalence (TYL= 63% vs. IGY= 52%; P = 0.40) or severity (P = 0.23). There was a greater proportion of erythromycin-resistant Enterococcus growth for TYL than IGY (42.6 vs. 21.9%; P < 0.04). Specifically formulated IGY feed additives may be viable alternatives to antibiotics in the diets of feedlot cattle.

81 Evaluation of feeding immunoglobulin-Y to Holstein feeder cattle for the control of liver abscesses & reduction of macrolide-resistant bacteria dissemination

Abstract Despite the regular use of macrolide-antibiotics in the diets of feedlot cattle, liver abscesses persist representing a financial burden to both pre- and post-mortem sectors of the beef industry. Alternative strategies, which simultaneously address liver infections and reduce the dissemination of antibiotic-resistant bacteria, are needed. An immunoglobulin-Y (IGY) product, developed to target Fusobacterium necrophorum andTrueperella pyogenes, was evaluated for the control of liver abscesses and its effect on macrolide-resistant Enterococcus growth from feces. Sixty-four Holstein steers (Initial BW= 384±13.5kg) consuming a finishing diet (90% concentrate) for 188 d (Final BW= 660±42 kg) were used in a completely randomized design where treatments included: TYL (Tylosin phosphate fed at 90 mg/hd/day; n = 32) or IGY (fed at 2.5g/hd/day; n = 32). Feed intake was recorded daily and BW was recorded every 28 d until slaughter. After 150 d on treatment fecal samples were randomly obtained from each treatment group four times (n = 40) and cultured on bile-esculine agar enriched with 8 μg/mL of erythromycin. Continuous variables of feedlot growth performance, carcass data and bacterial growth were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effects of steer or sampling day, respectively. Ordinal carcass data was analyzed using the FREQ procedure of SAS observing the Cochran-Mantel-Haenszel value. Treatment did not affect DMI (P = 0.21), ADG (P = 0.50) or G:F (P = 0.36). Treatment neither affected hot carcass weight (P = 0.37) nor longissimus muscle area (P = 0.25). However, TYL resulted in greater back fat thickness than IGY (0.32 vs. 0.23 in.; P < 0.02) and consequently greater numerical yield grade (P < 0.02). Quality grade was not affected by treatment (P = 0.65). No treatment difference was observed for liver abscesses prevalence (TYL= 63% vs. IGY= 52%; P = 0.40) or severity (P = 0.23). There was a greater proportion of erythromycin-resistant Enterococcus growth for TYL than IGY (42.6 vs. 21.9%; P < 0.04). Specifically formulated IGY feed additives may be viable alternatives to antibiotics in the diets of feedlot cattle.

83 Evaluation of feeding immunoglobulin-Y to Holstein feeder cattle for the control of liver abscesses & reduction of macrolide-resistant bacteria dissemination

Abstract Despite the regular use of macrolide-antibiotics in the diets of feedlot cattle, liver abscesses persist representing a financial burden to both pre- and post-mortem sectors of the beef industry. Alternative strategies, which simultaneously address liver infections and reduce the dissemination of antibiotic-resistant bacteria, are needed. An immunoglobulin-Y (IGY) product, developed to target Fusobacterium necrophorum and Trueperella pyogenes, was evaluated for the control of liver abscesses and its effect on macrolide-resistant Enterococcus growth from feces. Sixty-four Holstein steers (Initial BW= 384± 13.5kg) consuming a finishing diet (90% concentrate) for 188 days (Final BW= 660± 42 kg) were used in a completely randomized design where steer was experimental unit and treatments included: TYL (Tylosin phosphate fed at 90mg/hd/day; n = 32) or IGY (fed at 2.5g/hd/day; n= 32). Feed intake was recorded daily and BW was recorded every 28 days until slaughter. After 150 d on treatment fecal samples were randomly obtained from each treatment group four times (n = 40) and cultured on bile-esculine agar enriched with 8 μg/mL of erythromycin. Continuous variables of feedlot growth performance, carcass data and bacterial growth were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effects of steer or sampling day, respectively. Ordinal carcass data were analyzed using the FREQ procedure of SAS observing the Cochran-Mantel-Haenszel value. Treatment did not affect DMI (P= 0.21), ADG (P = 0.50) or G:F (P = 0.36). Treatment neither affected hot carcass weight (P = 0.37) nor longissimus muscle area (P = 0.25). However, TYL resulted in greater back fat thickness than IGY (0.32 vs. 0.23 in.; P < 0.02) and consequently greater numerical yield grade (P < 0.02). Quality grade was not affected by treatment (P = 0.65). No treatment difference was observed for liver abscesses prevalence (TYL=63% vs. IGY=52%; P = 0.40) or severity (P = 0.23). There was a greater proportion of erythromycin-resistant Enterococcus growth for TYL than IGY (42.6 vs. 21.9%; P < 0.04). Specifically formulated IGY feed additives may be viable alternatives to antibiotics in feedlot cattle diets.

Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model.

The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in 2012 and causes severe and often fatal acute respiratory illness in humans. No approved prophylactic and therapeutic interventions are currently available. In this study, we have developed egg yolk antibodies (immunoglobulin Y (IgY)) specific for MERS-CoV spike protein (S1) in order to evaluate their neutralizing efficiency against MERS-CoV infection. S1-specific immunoglobulins were produced by injecting chickens with purified recombinant S1 protein of MERS-CoV at a high titer (5.7 mg/mL egg yolk) at week 7 post immunization. Western blotting and immune-dot blot assays demonstrated that the IgY antibody specifically bound to the MERS-CoV S1 protein. Anti-S1 antibodies were also able to recognize MERS-COV inside cells, as demonstrated by an immunofluorescence assay. Plaque reduction and microneutralization assays showed the neutralization of MERS-COV in Vero cells by anti-S1 IgY antibodies and non-significantly reduced virus titers in the lungs of MERS-CoV-infected mice during early infection, with a nonsignificant decrease in weight loss. However, a statistically significant (p = 0.0196) quantitative reduction in viral antigen expression and marked reduction in inflammation were observed in lung tissue. Collectively, our data suggest that the anti-MERS-CoV S1 IgY could serve as a potential candidate for the passive treatment of MERS-CoV infection.

Innate and adaptive immune proteins in the preen gland secretions of male house sparrows

Recent studies have demonstrated that preen oil acts to reduce or eliminate feather‐associated bacteria. The mechanisms underlying this antibacterial activity, however, are incompletely understood. In addition to the activity of alcohols (i.e. 3,7‐dimethyloctan‐1‐ol), recent research has suggested that antimicrobial peptides may underlie the antibacterial activity of preen oil. Here, we document the presence of innate and adaptive immune proteins, lysozyme and immunoglobulin Y (IgY), in the preen oil of house sparrowsPasser domesticus. We suggest lysozyme functions as an antimicrobial agent, with potentially important impacts against Gram‐positive feather degrading bacteria. Furthermore, both lysozyme and IgY likely act in local immune defence of the preen gland, and may also play a role in regulating the local microbiome, with potentially important consequences for chemical communication and signalling. Our findings suggest that the preen gland and its secretions should be considered an integral part of the body's first line of defence against invading infections.

Generation of Chicken IgY against SARS-COV-2 Spike Protein and Epitope Mapping.

This new decade has started with a global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), precipitating a worldwide health crisis and economic downturn. Scientists and clinicians have been racing against time to find therapies for COVID-19. Repurposing approved drugs, developing vaccines and employing passive immunization are three major therapeutic approaches to fighting COVID-19. Chicken immunoglobulin Y (IgY) has the potential to be used as neutralizing antibody against respiratory infections, and its advantages include high avidity, low risk of adverse immune responses, and easy local delivery by intranasal administration. In this study, we raised antibody against the spike (S) protein of SARS-CoV-2 in chickens and extracted IgY (called IgY-S) from egg yolk. IgY-S exhibited high immunoreactivity against SARS-CoV-2 S, and by epitope mapping, we found five linear epitopes of IgY-S in SARS-CoV-2 S, two of which are cross-reactive with SARS-CoV S. Notably, epitope SIIAYTMSL, one of the identified epitopes, partially overlaps the S1/S2 cleavage region in SARS-CoV-2 S and is located on the surface of S trimer in 3D structure, close to the S1/S2 cleavage site. Thus, antibody binding at this location could physically block the access of proteolytic enzymes to S1/S2 cleavage site and thereby impede S1/S2 proteolytic cleavage, which is crucial to subsequent virus-cell membrane fusion and viral cell entry. Therefore, the feasibility of using IgY-S or epitope SIIAYTMS-specific IgY as neutralizing antibody for preventing or treating SARS-CoV-2 infection is worth exploring.

Avian anti-NS1 IgY antibodies neutralize dengue virus infection and protect against lethal dengue virus challenge

Dengue is the most prevalent arboviral disease in humans and a continually increasing global public health burden. To date, there are no approved antiviral therapies against dengue virus (DENV) and the only licensed vaccine, Dengvaxia, is exclusively indicated for individuals with prior DENV infection. Endothelial hyperpermeability and vascular leak, pathogenic hallmarks of severe dengue disease, can be directly triggered by DENV non-structural protein 1 (NS1). As such, anti-NS1 antibodies can prevent NS1-triggered endothelial dysfunction in vitro and pathogenesis in vivo. Recently, goose-derived anti-DENV immunoglobulin Y (IgY) antibodies were shown to neutralize DENV and Zika virus (ZIKV) infection without adverse effects, such as antibody-dependent enhancement (ADE). In this study, we used egg yolks from DENV-immunized geese to purify IgY antibodies specific to DENV NS1 epitopes. We determined that 2 anti-NS1 IgY antibodies, NS1-1 and NS1-8, were capable of neutralizing DENV infection in vitro. In addition, these antibodies did not cross-react with the DENV Envelope (E) protein nor enhance DENV or ZIKV infection in vitro. Intriguingly, NS1-8, but not NS1-1, partially blocked NS1-induced endothelial dysfunction in vitro while neither antibody blocked binding of soluble NS1 to cells. Finally, prophylactic treatment of mice with NS1-8 conferred significant protection against lethal DENV challenge. Although further research is needed to define the mechanism of action of these antibodies, our findings highlight the potential of anti-NS1 IgY as a promising prophylactic approach against DENV infection.

Application of IgY antibodies against staphylococcal protein A (SpA) of Staphylococcus aureus for detection and prophylactic functions.

In the present study, immunoglobulin Y (IgY) antibodies were raised in hens against the surface staphylococcal protein A (SpA) of Staphylococcus aureus. Anti-SpA IgY were tested in vitro for diagnostic applications, bacteriostatic, and biofilm inhibition effects. A specific and sensitive immunocapture PCR (IPCR) was developed to detect S. aureus from food, clinical, and environmental samples. Anti-SpA IgY were used for capturing S. aureus cells from different matrices. Chicken antibodies were chosen over mammalian antibodies based on its inertness to immunoglobulin (Ig)-binding property of SpA protein. No cross-reactivity was encountered with closely related Gram-positive and Gram-negative food pathogens. Inter-assay variation is < 10%. The assay was found suitable for testing on solid and liquid food samples, skin, and nasal swabs. The assay showed limit of detection of ≥ 102 CFU/mL from broth cultures and 102 to 103 CFU/ml from diverse natural samples. This assay overcomes the false positives commonly encountered while using mammalian immunoglobulins (IgG). Anti-SpA IgY antibodies were tested for their bacteriostatic effect on the growth of S. aureus. IgY antibodies at a concentration of 150 μg/ml inhibited the growth of S. aureus completely indicating the potential of IgY antibodies in neutralization of infectious pathogens. Similarly, anti-SpA IgY at MIC50 concentration reduced biofilm formation by ~ 45%. In view of advantages offered by IgY antibodies for specific detection of S. aureus in immunocapture PCR (IPCR) assay and in vitro neutralization potential of S. aureus, we recommend using IgY over conventional IgG of mammals involving S. aureus and its antigens. KEY POINTS: • IPCR with anti-SpA IgY for S. aureus was specific and sensitive for natural samples. • Anti-SpA IgY at 150 ug/ml displayed growth inhibition of S. aureus strains temporarily. • Anti-SpA IgY at MIC50 concentrations inhibited the biofilm formation partially.

THE POTENTIAL OF ADJUVANT AGAINST PRODUCTION OF ANTISTREPTOCOCCAL IMMUNOGLOBULIN Y (IGY) IN AQUACULTURE

This study was conducted to explore the potential of adjuvant for the production of immunoglobulin Y (IgY) as antistreptococcosis in layer chicken with mass production orientation. Enterococcus faecalis which causes streptococcosis in the red tilapia was selected as a candidateantigen. The production of immunoglobulin Y (IgY) was carried out on Isa Brown layer chickens and aged around 20 weeks. Furthermore, thechickens were grouped into four groups (A, B, C, and D groups), each consisting of three chickens based on the type of adjuvant, while twochickens were used as a control group. Each group was treated by giving MONTANIDE™ ISA 71R VG adjuvant (A), Freund's adjuvant (B), aluminum potassium sulphate adjuvant (KAl(SO4)2∙12H2O) concentration of 50 ppm in pH 7 (C), and only antigens without adjuvant (D). Chickens were kept for 35 days and each week was checked for presence the IgY antigen in the serum and egg yolk. Booster was conducted on 14th and 28th days of maintenance. The results showed that IgY in treatment group A was detected on day 28 in the serum and day 35 in the yolk. Whereas the treatment group B could be detected on day 35 in the serum. However, the IgY was not detected in the serum and yolk in C, D, and control groups until the end of the maintenance. Based on the results, it can be concluded that the appearance of IgY in serum and yolk in a relatively fast time is obtained in the combination of Enterococcus faecalis antigen with the emulsion of water-in-oil adjuvant (SEPPICMONTANIDE™ ISA 71R VG) compared to the other types of adjuvant that use in this study.

Formulation of alginate/carrageenan microgels to encapsulate, protect and release immunoglobulins: Egg Yolk IgY

In this study, alginate/κ-carrageenan hydrogel beads were developed to encapsulate and release egg yolk immunoglobulin Y (IgY) under simulated gastrointestinal conditions. The effects of κ-carrageenan concentration on the encapsulation and loading efficiency of the hydrogel beads was assessed, as well as on their swelling and release behavior in a simulated human gut. An κ-carrageenan concentration of 0.30% gave the optimum encapsulation and loading efficiency. After incubation in simulated gastric fluids, there was no loss in IgY activity when it was encapsulated within alginate/carrageenan beads but around 35% loss when it was encapsulated in pure alginate beads. This effect was attributed to a stronger electrostatic attraction and a more closely packed biopolymer network formed within the composite beads, based on measurements of their electrical and microstructural properties. The composite beads were resistant to swelling in the stomach phase, but not in the intestine phase. The kinetics of swelling under simulated gastric conditions was found to follow Schott's second-order model. The gastrointestinal digestion of the IgY was slower in the composite beads than in the pure alginate beads. The release kinetics of IgY from the composite beads was fitted by an irregular release model. Our results suggest that alginate/κ-carrageenan composite beads are efficient carriers for the delivery systems of hydrophilic immunoglobins.

Colorimetric immunoassay for rapid detection of Staphylococcus aureus based on etching-enhanced peroxidase-like catalytic activity of gold nanoparticles.

A novel colorimetric immunoassay for the detection of Staphylococcus aureus (S. aureus) based on a combination of immunomagnetic separation and signal amplification via etching-enhanced peroxidase-like catalytic activity of gold nanoparticles (AuNPs) was developed. Nanoconjugates composed of gold and iron oxide nanoparticles were synthesized and further modified with antiS. aureus immunoglobulin Y (IgY), which was used for the selective enrichment and rapid separation of target bacteria in complex matrices. AuNPs functionalized with antiS. aureus aptamer were used as an artificial enzyme which has peroxidase-like catalysis activity. Catalytic activity of AuNPs is inhibited by modifying aptamer. However, catalysis of modified AuNPs remarkably enhanced by hydrogen peroxide etching. Based on collecting unbound modified AuNPs in thesupernatant and 3,3',5,5'-tetramethylbenzidine-hydrogen peroxide reporting system, the yellow color of solution decreases linearly with increasing the concentration of S. aureus ranging from 10 to 106cfu/mL. The limit of detection is 10cfu/mL, and total detection time is 65min. The recoveries of the S. aureus spiked in food samples are 88.2-119.8%. Schematic illustration of colorimetric method for detection of S. aureus based on the IgY-Fe3O4/Au nanocomposites as capture probes and apt-AuNPs as artificial enzyme with etching-enhanced peroxidase-like catalytic activity.

Microencapsulation of immunoglobulin Y: optimization with response surface morphology and controlled release during simulated gastrointestinal digestion.

Immunoglobulin Y (IgY) is an effective orally administered antibody used to protect against various intestinal pathogens, but which cannot tolerate the acidic gastric environment. In this study, IgY was microencapsulated by alginate (ALG) and coated with chitooligosaccharide (COS). A response surface methodology was used to optimize the formulation, and a simulated gastrointestinal (GI) digestion (SGID) system to evaluate the controlled release of microencapsulated IgY. The microcapsule formulation was optimized as an ALG concentration of 1.56% (15.6 g/L), COS level of 0.61% (6.1 g/L), and IgY/ALG ratio of 62.44% (mass ratio). The microcapsules prepared following this formulation had an encapsulation efficiency of 65.19%, a loading capacity of 33.75%, and an average particle size of 588.75 μm. Under this optimum formulation, the coating of COS provided a less porous and more continuous microstructure by filling the cracks on the surface, and thus the GI release rate of encapsulated IgY was significantly reduced. The release of encapsulated IgY during simulated gastric and intestinal digestion well fitted the zero-order and first-order kinetics functions, respectively. The microcapsule also allowed the IgY to retain 84.37% immune-activity after 4 h simulated GI digestion, significantly higher than that for unprotected IgY (5.33%). This approach could provide an efficient way to preserve IgY and improve its performance in the GI tract.

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.

Immunoconjugates to increase photoinactivation of bovine alphaherpesvirus 1 in semen

Artificial insemination and in vitro embryo production are increasingly used to improve the reproductive efficiency of herds, however success of these techniques depends on the sanitary quality of the semen. Insemination centers commonly use antibiotics in their routine procedure, but they are not able against viruses. In this paper, we demonstrate a new approach for disinfecting virus in bovine semen using photoimmunoinactivation, an adaptation of the photodynamic inactivation (PDI) methodology. The photosensitizers (PSs), hematoporphyrin (HP) and zinc tetracarboxy-phthalocyanine (ZnPc) were conjugated to Immunoglobulin Y (IgY) anti-bovine alphaherpesvirus 1 (BoHV-1) and used for PDI against the BoHV-1 viruses in cell culture and compared to the unconjugated PSs. Both treatments proved to be efficient, but a significant decrease in the irradiation time required to completely eliminate the virus was observed in the samples treated with the immunoconjugates. Photophysical measurements help us to understand the coupling between PSs and IgY and the evaluated production of singlet oxygen. Following the cell culture test, the same approach was applied in semen artificially infected with BoHV-1. The immunoconjugates were also efficient for complete virus inactivation up to 5 min of irradiation and proved to be safe using several parameters of sperm viability, demonstrating the feasibility of our strategy for disinfection viruses in semen.

Conjugation and Characterization of Latex Particles with Toxoplasma gondii–specific Immunoglobulin Y Antibodies for Diagnostic Aim and Evaluation Efficiency in In Vitro Culture

Toxoplasma gondii is a parasite that causes severe health problems in the world. Toxoplasmosis, an infection caused by T. gondii, leads to high risk of mortality in patients with immunodeficiency, transplantation, and cancer. Besides that, it causes miscarriages in pregnancy, various abnormalities such as hydrocephalus in infants and congenital diseases. Because the clinical indication of the disease is not specific, it is confused with many diseases, and this leads to the necessity of directly detecting the presence of the toxoplasmosis. Therefore, various diagnostic assays are needed for the diagnosis of the disease. Amongs them, latex agglutination assay is widely used for the detection of specific antibodies or antigens in samples. Latex particles are coated with immunogenic molecules (antigens) to detect antibodies in the blood or used to identify antigens when coated with specific antibodies. In both, aggregation of latex particles results in agglutination. Monoclonal antibodies are often used in latex agglutination assay as in other diagnostic methods. However, monoclonal antibodies can be produced in low quantities at a high cost. Besides, to produce monoclonal antibodies, an experienced staff, a well-equipped cell culture laboratory, a long period of time, and a burdened budget are needed. In recent years, as an alternative to monoclonal antibodies, immunoglobulin Y (IgY) antibodies, which are obtained from chicken eggs, and specifically produced against desired antigenic constructs, have become quite attractive in terms of both low cost and abundant production without requiring infrastructure. In contrast, the latex assay based on IgY antibodies for use in the diagnosis of T. gondii has not been developed. This study aimed to conjugate T. gondii-specific IgY antibodies to latex particles, characterize the particles by Fourier transform infrared spectroscopy, scanning electron microscopy, and spectroscopic methods, and finally demonstrate the interaction with T.gondii parasites in culture with scanning electron microscopy analysis.