IgY Antibodies Research Articles

Assessing an O-antigen deficient, live attenuated Salmonella Gallinarium strain that is DIVA compatible, environmentally safe, and protects chickens against fowl typhoid

The objective of the present study was to create a highly attenuated, safe Salmonella Gallinarium (SG) vaccine strain for chicken vaccination against fowl typhoid (FT) diseases. The SG vaccine strain (SGVS) consists of three virulence-related gene deletions, namely, lon, cpxR, and rfaL. The parent strain (SGPS) with Δlon ΔcpxR genotype was utilized as the host strain for in-frame rfaL gene deletion by lambda red recombination. The SGVS was highly attenuated with improved environmental safety by zero fecal contamination beyond seven days for both oral and intramuscular immunization routes. Upon inoculation into 1-month-old young chicken, no vaccine-induced adverse behaviors were observed and did not cause a chronic state of infection as the SG wild-type strain did. Immunization of chicken elicited both humoral and cell-mediated immune responses demarcated by, IgY antibody assessment, T-cell responses in peripheral blood mononuclear cells, and the induction of immunomodulatory cytokines, IFN-γ, IL-2, IL-12, and IL-4 to resemble both Th1 and Th2 type of immune responses. The immunological assessment revealed a high level of efficacy of the SGVS when inoculated via the IM route than the oral route. The strain was less cytotoxic with reduced cytotoxicity on chicken macrophages and was DIVA capable with minimum reactivity of immunized serum with purified SG lipopolysaccharides. The challenge study could generate 70% protection in chicken for SGVS, whereas no birds were protected in the PBS challenged group. The protection levels were evident in histopathological assessment of spleen and liver specimens and also the external appearance of the spleen with reduced lesions on immunized groups. Further experiments may be warranted to dose and route optimization for further increase in the protection level derived by present SGVS.

Preclinical Assessment of IgY Antibodies Against Recombinant SARS-CoV-2 RBD Protein for Prophylaxis and Post-Infection Treatment of COVID-19.

Within the framework of the current COVID-19 pandemic, there is a race against time to find therapies for the outbreak to be controlled. Since vaccines are still tedious to develop and partially available for low-income countries, passive immunity based on egg-yolk antibodies (IgY) is presented as a suitable approach to preclude potential death of infected patients, based on its high specificity/avidity/production yield, cost-effective manufacture, and ease of administration. In the present study, IgY antibodies against a recombinant RBD protein of SARS-CoV-2 were produced in specific-pathogen-free chickens and purified from eggs using a biocompatible method. In vitro immunoreactivity was tested, finding high recognition and neutralization values. Safety was also demonstrated prior to efficacy evaluation, in which body weight, kinematics, and histopathological assessments of hamsters challenged with SARS-CoV-2 were performed, showing a protective effect administering IgY intranasally both as a prophylactic treatment or a post-infection treatment. The results of this study showed that intranasally delivered IgY has the potential to both aid in prevention and in overcoming COVID-19 infection, which should be very useful to control the advance of the current pandemic and the associated mortality.

Oral Immunization of Chickens with Probiotic Lactobacillus crispatus Constitutively Expressing the α-β2-ε-β1 Toxoids to Induce Protective Immunity.

Clostridium perfringens (C. perfringens) is a bacterium that commonly causes zoonotic disease. The pathogenicity of C. perfringens is a result of the combined action of α, β, and ε exotoxins. In this study, Lactobacillus crispatus (pPG-T7g10/L. crispatus) expressing the main toxoids of C. perfringens, α, ε, β1, and β2, with EGFP-labeling, was constructed, and the protective effect was estimated in chickens. The α-β2-ε-β1 toxoid was constitutively expressed for confirmation by laser confocal microscopy and western blotting, and its immunogenicity was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical assays. After booster immunization, the probiotic vaccine group showed significantly higher levels (p < 0.05) of specific secretory IgA (sIgA) and IgY antibodies in the serum and intestinal mucus. Furthermore, the levels of cytokines, including interferon (IFN)-γ, interleukin (lL)-2, IL-4, IL-10, IL-12, and IL-17, and the proliferation of spleen lymphocytes in chickens orally immunized with pPG-E-α-β2-ε-β1/L. crispatus increased significantly. Histopathological observations showed that the intestinal pathological changes in chickens immunized with pPG-E-α-β2ε-β1/L. crispatus were significantly alleviated. These data reveal that the probiotic vaccine could stimulate mucosal, cellular, and humoral immunity and provide an active defense against the toxins of C. perfringens, suggesting a promising candidate for oral vaccines against C. perfringens.

A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45–UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.

Production of Functional Ice Cream Fortified by Immunoglobulin Y against Escherichia coli O157:H7 and Helicobacter Pylori

The production of effective functional foods that can increase public health is paramount to scientists. This study aimed to optimize methods for developing bio-ice cream comprising IgY versus Escherichia coli O157 and Helicobacter pylori. Leghorn hens were immunized intramuscularly with antigens. The egg yolk was separated from the egg, and the IgY antibody was then purified using the PEG 6000 method. Maximum IgY was reached in 2 weeks with IgY concentrations of 14.56, 11.46, 16.34, and 6.87 mg/ml for H. pylori, E. coli O157, E. coli + H. pylori, and control, respectively. The results showed that 0.6 mg/ml concentration of IgY had a significant inhibitory effect on bacteria growth using disk diffusion methods. Also, ELISA results showed that IgY activity was reliable and remained active in ice cream for 3 months. According to sensory evaluation, the global acceptance scores and the other attributes revealed the acceptable acceptance of IgY egg yolk ice cream. Consequently, it can be stated that the ice cream matrix can be considered an ideal candidate for carrying antibodies into the host body, as the specific ingredients of the ice cream matrix can appropriately protect IgY antibodies.

A new approach of immunotherapy against Crotalus snakes envenoming: ostrich (Struthio camelus) egg yolk antibodies (IgY-technology)

Crotalid envenomation is a neglected collective health problem involving many countries in America, which need secure and inexpensive snake anti-venom treatments. Here, high antibody titers (IgY) were raised in the Ostrich (Struthio camelus) egg yolk by immunizing with the venom of Venezuelan venomous Crotalus snakes. Ostriches were immunized with a pool of venoms from common rattlesnake (Crotalus durissus cumanensis), Uracoan rattlesnake (Crotalus vegrandis), Guayana rattlesnake (Crotalus durissus ruruima) and black rattlesnake (Crotalus pifanorum). The anti-snake venom antibodies were prepared from egg yolk by the water dilution method, enriched by the addition of caprylic acid (CA) and precipitation with ammonium sulfate at 30% (W/V). The purity and molecular mass of the final product was satisfactory, yielding a single ∼ 175 kDa band in SDS-PAGE gels ran under non-reducing conditions. In the immunoblot analysis, specific binding of the antivenom was observed with most venom proteins. The LD50 was 16.5 g/mouse (825 µg/kg body weight). High titers of IgY against Crot/pool venom were shown by ELISA. The median effective dose (ED50) was 19.66 mg/2LD50. IgY antibodies neutralized efficiently the Crot/pool venom lethality. As far as we know, this is the first anti-snake venom produced in ostriches, which could make this technology an affordable alternative for low-income countries, since it is likely to produce about 2-4 g of IgY per ostrich egg. Hence, almost 400 g of IgY can be purified from only one ostrich during a year. In addition, there are enormous differences in the cost of investment in the maintenance of horses, from the points of view of infrastructure, feeding and veterinary care, in which the cost can reach USD 100 per animal per day, compared to a maintenance cost of USD 146 per month per producing bird. These results are encouraging and could easily be extrapolated to the manufacturing of other antivenoms and antitoxins as well, as they could be applied to the manufacturing of potential diagnostic tools.

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5–1 and rH5–2, respectively. The systemic prime and boost immunizations proved that rH5–1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5–2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5–1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5–2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5–1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

Affordable IgY-based antiviral prophylaxis for resource-limited settings to address epidemic and pandemic risks.

BackgroundThe COVID-19 pandemic caused by SARS-CoV-2 exposed a global problem, as highly effective vaccines are challenging to produce and distribute, particularly in regions with limited resources and funding. As an alternative, immunoglobulins produced in eggs of immunized hens (IgY) can be a simple and inexpensive source for a topical and temporary prophylaxis. Here, we developed a method to extract and purify IgY antibodies from egg yolks of hens immunized against viral pathogen-derived proteins using low-cost, readily available materials, for use in resource-limited settings.MethodsExisting protocols for IgY purification and equipment were modified, including extraction from yolks and separation of water-soluble IgY using common household reagents and tools. A replacement for a commercial centrifuge was developed, using a home food processor equipped with a 3D printed adapter to enable IgY precipitation. IgY purification was verified using standard gel electrophoresis and Western blot analyses.ResultsWe developed a step-by-step protocol for IgY purification for two settings in low- and middle-income countries (LMIC): a local laboratory, where commercial centrifuges are available, or a more rural setting, where an alternative for expensive centrifuges can be used. Gel electrophoresis and Western blot analyses confirmed that the method produced highly enriched IgY preparation; each commercial egg produced ~ 90 mg of IgY. We also designed a kit for IgY production in these two settings and provided a cost estimate of the kit.ConclusionIgY purified from eggs of immunized local hens can offer a fast and affordable prophylaxis, provided that purification can be performed in a resource-limited setting. Here, we created a low-cost method that can be used anywhere where electricity is available using inexpensive, readily available materials in place of costly, specialized laboratory equipment and chemicals. This procedure can readily be used now to make an anti-SARS-CoV-2 prophylaxis in areas where vaccines are unavailable, and can be modified to combat future threats from viral epidemics and pandemics.

Specific anti-SARS-CoV-2 S1 IgY-scFv is a promising tool for recognition of the virus

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread globally, a series of vaccines, antibodies and drugs have been developed to combat coronavirus disease 2019 (COVID-19). High specific antibodies are powerful tool for the development of immunoassay and providing passive immunotherapy against SARS-CoV-2 and expected with large scale production. SARS-CoV-2 S1 protein was expressed in E. coli BL21 and purified by immobilized metal affinity chromatography, as antigen used to immunize hens, the specific IgY antibodies were extracted form egg yolk by PEG-6000 precipitation, and the titer of anti-S1 IgY antibody reached 1:10,000. IgY single chain variable fragment antibody (IgY-scFv) was generated by using phage display technology and the IgY-scFv showed high binding sensitivity and capacity to S1 protein of SARS-CoV-2, and the minimum detectable antigen S1 protein concentration was 6 ng/µL. The docking study showed that the multiple epitopes on the IgY-scFv interacted with multiple residues on SARS-CoV-2 S1 RBD to form hydrogen bonds. This preliminary study suggests that IgY and IgY-scFv are suitable candidates for the development of immunoassay and passive immunotherapy for COVID-19 to humans and animals.

Optimized method for the extraction of contaminant-free IgY antibodies from egg yolk using PEG 6000

Hens are oviparous vertebrates and produce IgY antibodies, which is the main type of immunoglobulin in the egg yolk, and high concentrations can be obtained by using a simple method that does not require sophisticated equipment and reagents. The Polyethylene Glycol 6000 method allows the removal of lipids and the precipitation of IgY in two days with an approximated purity of around 80%, however during the original protocol other contaminant proteins can be precipitated. To overcome the issue of contamination with other proteins and extraction time, we optimized the previously method described by Pauly et al. (2011) by adding some changes that improved the aforementioned problems.• Our protocol is customized by the addition of one more filtration step or one more step with PEG 6000 at 3.5% to avoid the contamination with lipids.• Additionally, the changes in the type of agitation, centrifugation and the skip of dialysis make the method more accessible for all the laboratories.• In summary, these modifications serve to enhance the purity, reduce the time for IgY extraction from egg yolk and make it more accessible for every basic research laboratory.

The possibility of using Ig Y-antibodies in immunotherapy

The importance of yolk antibodies (IgY) for their use in the diagnosis and treatment of human diseases is shown. The structural features of IgY compared to human immunoglobulins were studied. The disadvantages of obtaining hyperimmune sera from mammalian animals were revealed. A feature of IgY is its resistance to trypsin and chymotrypsin, which can contribute to more effective treatment of diseases. Data on optimization of the technology for obtaining yolk antibodies (IgY) from various bird species are presented. It is shown that 25-50 mg of highly purified immunoglobulin can be obtained from one bird egg. Obtaining immunoglobulins from eggs is more humane than using mammals for this purpose. Compared to ion exchange chromatography, gel chromatography, and ultrafiltration, the methods of precipitation with various chemicals (ammonium sulfate) were found to be simpler in terms of methodology. The results of electrophoresis revealed two types of proteins with a molecular weight of about 65 kDa and 30 kDa, which corresponds to the mass of the «heavy» and «light» IgY chains. Quail species Coturnix coturnix japonica as a producer of biological raw materials for the production of diagnostic immunoglobulins have great advantages over chickens (Gallus domesticus).

Electrochemical immunosensor for detection of Plasmodium vivax lactate dehydrogenase.

BACKGROUND Malaria is a disease that affects many tropical and subtropical countries, including Brazil. The use of tests for malaria detection is one of the fundamental strategies recommended by the World Health Organization for the control and eradication of the disease. The lack of diagnostic tests leads to an increase in transmission and non-reporting cases.OBJECTIVES This work described an electrochemical immunosensor for detecting Plasmodium vivax lactate dehydrogenase antigen (Ag-PvLDH).METHODS The device has developed by immobilising egg yolk IgY antibodies (Ab-PvLDH) on a gold electrode surface using cysteamine as linker. The immunosensor fabrication was followed by differential pulse voltammetry, and contact angle measurements were performed to characterise the modified gold electrode surface.FINDINGS The results for Ag-PvLDH determination exhibit a linear response at 10-50 µg mL-1 concentration range, with a limit of detection of 455 ng mL-1. The excellent selectivity of the device was confirmed.MAIN CONCLUSIONS The developed immunosensor showed a good performance, therefore, it can be considered an alternative test to detect malaria caused by P. vivax.

Protection against avian coronavirus conferred by oral vaccination with live bacteria secreting LTB-fused viral proteins

The devastating impact of infectious bronchitis (IB) triggered by the IB virus (IBV), on poultry farms is generally curbed by livestock vaccination with live attenuated or inactivated vaccines. Yet, this approach is challenged by continuously emerging variants and by time limitations of vaccine preparation techniques. This work describes the design and evaluation of an anti-IBV vaccine comprised of E. coli expressing and secreting viral spike 1 subunit (S1) and nucleocapsid N-terminus and C-terminus polypeptides fused to heat-labile enterotoxin B (LTB) (LS1, LNN, LNC, respectively). Following chicken oral vaccination, anti-IBV IgY levels and cellular-mediated immunity as well as protection against virulent IBV challenge, were evaluated 14 days following the booster dose. Oral vaccination induced IgY levels that exceeded those measured following vaccination with each component separately. Following exposure to inactivated IBV, splenocytes isolated from chicks orally vaccinated with LNN or LNC -expressing bacteria, showed a higher percentage of CD8+ cells as compared to splenocytes isolated from chicks vaccinated with wild type or LTB-secreting E. coli and to chicks subcutaneously vaccinated. Significant reduction in viral load and percent of shedders in the vaccinated chicks was evident starting 3 days following challenge with 107.5 EID50/ml virulent IBV. Taken together, orally delivered LTB-fused IBV polypeptide-expressing bacteria induced virus-specific IgY antibody production and was associated with significantly shorter viral shedding on challenge with a live IBV. The proposed vaccine design and delivery route promise an effective and rapidly adaptable means of protecting poultry farms from devastating IB outbreaks.

Multi-functional magnetic molecular imprinting probe for visual detection of IgY antibodies.

Egg yolk antibodies (IgY)are analternative to mammalian antibodies, which have been successfully applied in treatment ofdisease, asimmunochemical reagents and asfood additives. A fast, accurate, and easy-to-operate detection method for IgY antibodies is needed. Herein, we developed a rapid and cost-effective colorimetric assay for the ultrasensitive detection of anti-S. aureus IgY antibodies using multi-functional magnetic molecularly imprinted polymers (MMIPs). The prepared MMIPs can recognize, adsorb, and separate the analyte from the matrix efficiently, and oxidize TMB to generate a colorimetric signal within ~ 60min. As low as 0.013mg·mL-1 was able to be detected by using this method. The visual detection limit reached 0.02mg·mL-1. By testing the IgY in milk samples, the feasibility was verified.

Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilities.

Chlamydial infections, caused by a group of obligate, intracellular, gram-negative bacteria, have health implications for animals and humans. Due to their highly infectious nature and zoonotic potential, staff at wildlife rehabilitation centers should be educated on the clinical manifestations, prevalence, and risk factors associated with Chlamydia spp. infections in raptors. The objectives of this study were to document the prevalence of chlamydial DNA shedding and anti-chlamydial antibodies in raptors admitted to five wildlife rehabilitation centers in California over a one-year period. Chlamydial prevalence was estimated in raptors for each center and potential risk factors associated with infection were evaluated, including location, species, season, and age class. Plasma samples and conjunctiva/choana/cloaca swabs were collected for serology and qPCR from a subset of 263 birds of prey, representing 18 species. Serologic assays identified both anti-C. buteonis IgM and anti-chlamydial IgY antibodies. Chlamydial DNA and anti-chlamydial antibodies were detected in 4.18% (11/263) and 3.14% (6/191) of patients, respectively. Chamydial DNA was identified in raptors from the families Accipitridae and Strigidae while anti-C.buteonis IgM was identified in birds identified in Accipitridae, Falconidae, Strigidae, and Cathartidae. Two of the chlamydial DNA positive birds (one Swainson's hawk (Buteo swainsoni) and one red-tailed hawk (Buteo jamaicensis)) were necropsied, and tissues were collected for culture. Sequencing of the cultured elementary bodies revealed a chlamydial DNA sequence with 99.97% average nucleotide identity to the recently described Chlamydia buteonis. Spatial clusters of seropositive raptors and raptors positive for chlamydial DNA were detected in northern California. Infections were most prevalent during the winter season. Furthermore, while the proportion of raptors testing positive for chlamydial DNA was similar across age classes, seroprevalence was highest in adults. This study questions the current knowledge on C. buteonis host range and highlights the importance of further studies to evaluate the diversity and epidemiology of Chlamydia spp. infecting raptor populations.

METABOLIC PROFILE IN HENS SUBJECT OF LONG TERM HYPERIMMUNIZATION

In the present paper, the metabolic profile was analysed in a group of adult Rhode Island hens extensively required for the production of IgY antibodies against bacterial entities. The results reveal changes in the leukocyte formula dominated by an increase in the percentage of heterophils and a decrease in the percentage of lymphocytes in the blood. An increase in the level of serum proteins with predominance of globulins is added to this. Increased uric acid levels were found also. Increased levels of GOT and GPT transaminases and -GT indicated an intensification of the protein metabolism, LDH and PA were not significantly modified. Regarding carbohydrate metabolism, it was found that serum glucose and lactate were maintained within physiological limits. The lipid profile was characterized by maintaining serum triglycerides and cholesterol within normal limits. Significant decrease in HDL and increase in LDL were interpreted as a decrease in the proteosynthetic capacity of the liver. The mineral profile was dominated by the increase of the level of sodium, iron, magnesium and calcium but by the decrease of the level of chlorine, potassium and phosphorus.

Production and purification of polyclonal anti-chicken IgY immunoglobulins in Goats and conjugation with Horseradish Peroxidase

Antibodies are very important components in medical diagnostics and various biochemical assays for diagnosis of the diseases. IgY is an immunoglobulin largely present in Avians. Anti-chicken IgY immunoglobulins conjugated with gold nanoparticles are used to diagnose the chicken infectious diseases by lateral flow assay (LFA) and enzyme linked immunosorbent assay (ELISA) immunodiagnostic tests. The main aim of this study is the production of antichicken IgY antibodies in goats, purification of antiantibody and conjugation with horse radish peroxidase (HRP) enzyme. The antibody was purified using protein G sepharose affinity column and the purity of the antibody was confirmed by SDS-PAGE analysis and by spectrophotometer. The purified anti IgY antibody was also evaluated by ELISA. The optimum dilution of prepared HRP conjugated anti IgY was 1:9600. The sensitivity and specificity of the antibody were evaluated and have no cross reactivity with other IgG antibodies. This study showed that protein G affinity purification could be best useful method for purification of polyclonal IgG antibodies.

Anti-flagellin IgY antibodies protect against Pseudomonas aeruginosa infection in both acute pneumonia and burn wound murine models in a non-type-specific mode

Pseudomonas aeruginosa (PA) is one of the most dominant causes of nosocomial infections in burn patients. Increasing emergence of antibiotic-resistant strains highlights the need for novel antimicrobial agents. Flagellin, the main component protein of flagellum, is determined as the major antigen interacting with anti-P. aeruginosa IgY antibodies. The current study was aimed to evaluate the antibacterial potency of IgY antibodies raised against recombinant type A, and B flagellins. The immunogenicity and specificity of IgY antibodies were confirmed through indirect ELISA and western blot analysis, respectively. Anti-flagellin IgYs reduced the motility, biofilm formation and invasion potency of both strains. The cell surface hydrophobicity (CSH) of bacteria was increased upon IgY treatment, and in vitro opsonophagocytosis assay confirmed the high protective potency of specific antibodies via polymorphonuclear leukocyte (PMN)-augmented bacterial cell killing. The protective efficacy of IgYs was also studied in both acute pneumonia and burn wound murine models. Anti-flagellin B-IgY induced 100 % and 40 % protection against laboratory, and hospital strains in burn wound model, respectively. Protection in acute pneumonia against all strains was 100 %. Anti-flagellin A-IgY failed to protect mice in burn wound model, but provided 100 % protection against all strains in acute pneumonia challenge. In vitro, ex vivo and in vivo experiments confirmed the dose-dependent and non-type specific essence of anti-flagellin IgY antibodies, providing the benefit of covering all strain types in a dose dependent manner. Our findings provide evidence that anti-flagellin IgY antibodies qualify as novel economical therapeutic option against PA infection.

Immunotherapeutic Efficacy of IgY Antibodies Targeting the Full-Length Spike Protein in an Animal Model of Middle East Respiratory Syndrome Coronavirus Infection.

Identified in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and often fatal acute respiratory illness in humans. No approved prophylactic or therapeutic interventions are currently available. In this study, we developed chicken egg yolk antibodies (IgY Abs) specific to the MERS-CoV spike (S) protein and evaluated their neutralizing efficiency against MERS-CoV infection. S-specific IgY Abs were produced by injecting chickens with the purified recombinant S protein of MERS-CoV at a high titer (4.4 mg/mL per egg yolk) at week 7 post immunization. Western blotting and immune-dot blot assays demonstrated specific binding to the MERS-CoV S protein. In vitro neutralization of the generated IgY Abs against MERS-CoV was evaluated and showed a 50% neutralizing concentration of 51.42 μg/mL. In vivo testing using a human-transgenic mouse model showed a reduction of viral antigen positive cells in treated mice, compared to the adjuvant-only controls. Moreover, the lung cells of the treated mice showed significantly reduced inflammation, compared to the controls. Our results show efficient neutralization of MERS-CoV infection both in vitro and in vivo using S-specific IgY Abs. Clinical trials are needed to evaluate the efficiency of the IgY Abs in camels and humans.

In vitro Inhibitory Activity of IgY Antibodies Against Salmonella Ser. Newport Isolated from Horses

Equine salmonellosis is caused by several Salmonella serotypes, including Salmonella Newport, which cause enterocolitis and diarrhea. Treatment usually includes the administration of antibiotics. However, since multidrug-resistant Salmonella is commonly detected, alternative options to control the pathogen are needed. One of these options is the use of specific egg yolk antibodies (IgY) for passive immunotherapy. Thus, the aim of our work was to produce IgY antibodies against an equine S. Newport strain and assess their in vitro inhibitory activity. To this end, laying hens were immunized with an inactivated S. Newport strain by using either Freund's or Montanide adjuvant and egg yolk extracts were obtained. The levels of specific IgY antibodies against Salmonella in sera and egg extracts were determined by dot-blot and microagglutination. Besides, the IgY extracts were characterized by total protein analysis, SDS-PAGE, Western Blot, and inhibition of bacterial motility. IgY extracts showed high purity (87.7 to 91.8 %), high microagglutination titers, and the ability to inhibit the motility of the bacterium. The results using Montanide were similar to those using the traditional Freund's adjuvant. Thus, Montanide may also be a good adjuvant to produce IgY. IgY-technology represents a potential tool for the control of salmonellosis in horses.