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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 discovery of antimicrobials is an outstanding achievement of mankind that led to the development of modern medicine. However, increasing antimicrobial resistance observed worldwide is rendering commercially available antimicrobials ineffective. This problem results from the bacterial ability to adapt to selective pressure, leading to the development or acquisition of multiple types of resistance mechanisms that can severely affect the efficacy of antimicrobials. The misuse, over-prescription, and poor treatment adherence by patients are factors strongly aggravating this issue, with an epidemic of infections untreatable by first-line therapies occurring over decades. Alternatives are required to tackle this problem, and immunotherapies are emerging as pathogen-specific and nonresistance-generating alternatives to antimicrobials. In this work, four types of antibody formats and their potential for the development of antibody-based immunotherapies against bacteria are discussed. These antibody isotypes include conventional mammalian polyclonal antibodies that are used for the neutralization of toxins; conventional mammalian monoclonal antibodies that currently have 100 IgG mAbs approved for therapeutic use; immunoglobulin Y found in birds and an excellent source of high-quality polyclonal antibodies able to be purified noninvasively from egg yolks; and single domain antibodies (also known as nanobodies), a recently discovered antibody format (found in camelids and nurse sharks) that allows for a low-cost synthesis in microbial systems, access to hidden or hard-to-reach epitopes, and exhibits a high modularity for the development of complex structures.

Adulteration of meat products is a serious problem in the modern society. Consumption of falsified meat products can be hazardous to health and/or lead to violating religious dietary principles. To identify such products, rapid and simple test systems for point-of-need detection are in demand along with complex laboratory methods. This study presents the first double lateral flow (immunochromatographic) test system, which allows simultaneous revealing two prevalent types of falsifications—undeclared addition of pork and chicken components to meat products. In the proposed test system, porcine myoglobin (MG) and chicken immunoglobulin Y (IgY) were used as specific biomarkers recognizable by antibodies. Within the optimization of the analysis, the concentrations of the immune reagents and regimes of their application on the working membrane were selected, which provided minimal limits of detection (LODs) for both analytes. The developed test system enables the detection of MG and IgY with the LODs of 10 and 12 ng/mL, respectively, which accords to addition of 0.1% of the undeclared meat compounds. The applicability of the test system to control the composition of raw meat mixtures and cooked food products was confirmed. The developed approach can be considered as a promising tool for monitoring composition of meat products.

Objective: Egg yolk immunoglobulin (IgY) is an antibiotic alternative to prevent and fight intestinal pathogenic infections. This study aimed to investigate the effects of sodium alginate/chitosan/sodium alginate IgY microcapsules on the growth performance, serum parameters, and intestinal health of broiler chickens.Methods: One-day-old broilers (Ross 308) were divided into five treatments, each with 10 replicates of five chickens. The dietary treatments were maintained for 28 days and consisted of a basal diet (NC), basal diet + 500 mg chlortetracycline/kg diet (CH), basal diet + 50 mg non-microencapsulated IgY/kg diet (NM), basal diet + 600 mg low levels microencapsulated IgY/kg diet (LM), and basal diet + 700 mg high levels microencapsulated IgY/kg diet (HM).Results: Throughout the 28-day trial period, the NM, LM, HM, and CH groups increased average daily gain compared with the NC group (p<0.05), and the HM group reduced feed conversion ratio compared with the CH group (p<0.05). The LM and HM groups increased relative organ weights of thymus and spleen compared with the CH and NM groups (p<0.05). The HM group improved the duodenal, jejunal and ileum villi height (VH) and villus height to crypt depth ratio (VH:CD) compared with the CH and NM groups (p<0.05). Compared with the CH group, the HM group increased serum immunoglobulin (IgA), immunoglobulin G (IgG), superoxide dismutase, total antioxidant capacity, and glutathione peroxidase levels (p<0.05), and decreased serum malondialdehyde levels (p<0.05). Compared with the NC group, the NM, LM, HM, and CH groups reduced colonic Escherichia coli and Salmonella levels (p<0.05). and the HM group promoted the levels of lactic acid bacteria and bifidobacteria compared with the CH group (p<0.05).Conclusion: Microencapsulation could be considered as a way to improve the efficiency of IgY. The 700 mg high levels microencapsulated IgY/kg diet could potentially be used as an alternative to antibiotics to improve the immune performance and intestinal health, leading to better performance of broiler chickens.

Broiler chickens are the highest source to fulfill animal protein consumption of the Indonesian people. The population of this type of chicken is very high because it does not require a large area in maintenance, has fast growth and good feed efficiency compared to other types of broiler poultry. Broiler farms generally use antibiotics as feed additives to trigger growth, increase body weight and improve the efficiency of food converted into meat. Antibiotics are also used for disease prevention and treatment, especially by commercial chicken farmers. Antibiotics used in livestock production account for about two-thirds of global antibiotic sales and consumption. The unwise use of antibiotics also increases the occurrence of antimicrobial resistance, especially in bacteria. This research goals to analyze the sustainability of using IgY affixes as a substitute for antibiotics in chicken feed. The results of the analysis are grouped based on the value of the sustainability level. The results of Multi Dimensional Scaling (MDS) analysis using the RAPFISH (Rapid Appraisal for Fisheries) analysis tool showed that the level of sustainability of IgY use was 66.49% with a quite sustainable category.

Norovirus is a leading cause of epidemic acute gastroenteritis worldwide, associated with significant morbidity, mortality, and economic loss. Despite its global impact, no licensed vaccine is currently available, and vaccine development remains challenging. We explored avian immunoglobulin Y (IgY) antibodies as a low-cost countermeasure against norovirus infection. We generated recombinant protruding (P) domain proteins from the capsid protein (VP1) of noroviruses, representing two GII.4 variants and the GII.6 genotype. These were combined into a single immunogen to immunize laying hens to produce norovirus VP1-specific IgY antibodies. Immunization of laying hens with the P domain proteins elicited high-titer (>1:450,000) P domain-specific IgY antibodies. The yolk-derived IgY effectively inhibited binding of various norovirus P particles to their histo-blood group antigen ligands, with 50% blocking titers (BT50) up to 1:8533 against homotypic GII.4 and 1:667 against heterotypic G1.1 Norwalk virus P particles. Importantly, the IgY neutralized replication of GII.4 norovirus in the human intestinal enteroid (HIE) system at a high titer of over 1:2500, equivalent to 0.70 µg/mL of total IgY. We also produced norovirus shell (S) domain proteins and corresponding IgY antibodies, which neutralized GII.4 norovirus replication in the HIE model at a titer of ~1:800, equivalent to 2.98 µg/mL of total IgY. This provides the first evidence that the S domain contains neutralizing epitopes. Our findings support the potential of IgY targeting norovirus P or S domains as a scalable, cost-effective strategy for preventing norovirus infection and disease.

Ciprofloxacin is metabolized from enrofloxacin for use in poultry to manage respiratory and gastrointestinal diseases, raising concerns due to its widespread tissue distribution and prolonged systemic persistence. This lateral flow immunoassay was designed to detect ciprofloxacin using an alternative IgY antibody binded with gold nanoparticles to detect ciprofloxacin residue in raw pork meat samples. The developed strip test achieved adequate sensitivity and specificity under the optimized conditions for pH, which is 7.8, and 20% of MeOH in 0.01 M phosphate buffer containing 1% Tween-20 was used for the buffer composition. An antibody concentration of 1.25 µg/mL was used to bind with gold nanoparticles as a probe for detection. The concentration of the test line (coating antigen) and control line (anti-IgY secondary antibody) was 0.5 mg/mL and 0.2 mg/mL, respectively. The efficiency of the developed strip test showed sensitivity with a 50% inhibitory concentration (IC50) of ciprofloxacin at 7.36 µg/mL, and the limit of detection was 0.2 µg/mL. The proposed strategy exhibited potential for monitoring ciprofloxacin in raw pork samples.

The aim of this study was to using purified chicken antibody (IgY) for developing solid phase competitive (SPC) enzyme-linked immunosorbent assay (ELISA) to detect the foot-and-mouth disease virus (FMDV) A serotype. After immunization of chickens, polyclonal immunoglobulin (IgY) antibodies were extracted and purified from egg yolk and yield was about 5.00 mg mL-1 of yolk as well as near 0.40 mg mL-1 of specific IgY antibody against FMDV serotype A. Also, optimized sucrose density gradient method produced 228 µg mL-1 whole virus which is much higher than that of the conventional method of sucrose density gradient method. The optimum concentration of purified capture IgY and bind type A antigen were 0.50 µg and 0.10 µg per well, respectively. The OD values < 0.70 were considered positive, and values ≥ 0.70 were negative for in-house kit base on standard controls. Statistical analysis base on 80 serum samples showed the 96.66% sensitivity, 100% specificity, 100% positive predictive value, 90.90% negative predictive value, 97.50% accuracy, and 98.33% reliability for serum samples for two commercial and in-house kits. The SPCE developed based on IgY antibody is a suitable alternative for the detection of antibodies after vaccination against type A FMDV with high sensitivity and specificity. The present research demonstrated the possibility of commercial development of the SPCE kit using IgY antibodies for the detection of FMDV antibodies in serum samples with adequate sensitivity and accuracy.

Immunoglobulin Y (IgY) is the predominant antibody found in hen’s ( Gallus domesticus ) egg yolk. This antibody, developed against several microorganisms in hen egg yolk, has been successfully used as an alternative to immunoglobulins from mammals for use in immunodiagnostics and immunotherapy. Enteropathogenic Escherichia coli (E.coli) F4 is the main etiological agent associated with swine neonatal diarrhea, and it causes notable economic losses in swine production. The aim of the present study was to evaluate the relationship between humoral immune response and the activation of gut-associated lymphoid tissue (GALT) in laying hens intramuscularly immunized with E. coli F4. Adult laying Shaver hens were immunized with a bacterin based on an inactivated lysate E. coli F4 strain that was originally isolated from neonatal piglet diarrhea, following a recommended schedule. The percentage of B lymphocytes in blood and spleen homogenates was determined by flow cytometry. Villi histomorphometry and the size of germinal centers (GC) activated in GALT and the spleen were measured in histological samples either stained with hematoxylin/eosin or through immunofluorescence. Antibody and isotype-specific antibodies in serum and egg yolk were measured using indirect enzyme-linked immunosorbent assay (ELISA). Secretory and serum immunoglobulin A (IgA) were measured by ELISA tests. Laying hen with intramuscular immunization with E. coli F4 lysate, activated both mucosal and systemic protection. Mucosal protection was provided through B lymphocytes, and most of them were activated on Peyer’s patches and esophageal tonsils, in GALT. Furthermore, increased B lymphocyte number in the lamina propria of the gut, and increased intraepithelial plasmatic cell number, produced high levels of mucosal IgA. Activated B lymphocytes interacted with absorptive cells, immune cells, and microbiota in the gut, producing signals that were translated into a powerful physical defense by producing a greater volume of mucin from an increased number of goblet cells. Systemic protection was provided through B lymphocyte activation of spleen GC, which produced hugely specific IgY serum levels. One week later, this specific IgY was deposited in the yolk. This suggests that GALT is a key immunologic tissue inside the mucosal immune system, acting as the “command center” for humoral reaction.

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.