Chicken Egg Yolk Immunoglobulin Research Articles

Development of chicken egg yolk antibodies (IgY) against venom of cobra (Naja naja) and krait (Bungarus caeruleus) and a study on its neutralization potential

The Big Four snakes are the leading causes of snakebite mortality in India. Among them, The Indian spectacled cobra (Naja naja) and common krait (Bungarus caeruleus) contribute about one-third of the deaths. The conventional anti-snake venoms (ASV) produced from equine serum are found to be expensive, with a high frequency of clinical side effects, low specificity, and production drawbacks. The current study, therefore, attempted to develop chicken egg yolk immunoglobulins (IgY) as antivenoms against the venoms of cobra and krait venoms. The major pathophysiological properties of the venoms and the efficacy of monovalent ASV IgY were evaluated. The titer of anti-cobra and anti-krait IgY was found to be 1:64000 on the 145th day post-immunization. The results showed that venoms (lethal doses (LD50): cobra:0.375 mg/kg, krait:0.0375 mg/kg of mice) had hemolytic, pro-coagulant, inflammatory, hemorrhagic, and edematic activities that were effectively neutralized by ASV-IgY. 1 mL of monovalent ASV IgY was able to neutralize 0.22 mg of cobra venom and 0.028 mg of krait venom whereas 1 mL of commercial polyvalent antivenom was able to neutralize 0.28 mg of cobra venom and 0.035 mg of krait venom. The study results suggest that using chicken IgY as an monovalent antivenom could effectively treat snakebites.

The efficacy of a chicken antibody for the development of immunoassay‐based rapid detection in sugarcane mosaic virus disease

Sugarcane Mosaic Virus (SCMV) infection is one of the most serious problems that can result in severe yield loss of sugarcane. Since the symptoms of SCMV infection are similar to other biotic and abiotic stress symptoms, the development of a rapid diagnostic with high precision is required. The use of laboratory animals such as rabbits is required for antibody production in immunoassay‐based detection. However, due to its many advantages, specific chicken egg yolk immunoglobulin (IgY) has received considerable attention as an alternative antibody production in immunodiagnostics for infectious diseases. In this study, IgY antibody against SCMV recombinant coat protein (CP) was successfully obtained from chicken blood serum and tested to compare its efficacy against antibody from rabbit (IgG) using immunocapture reverse transcription‐polymerase chain reaction (IC‐RT‐PCR). The result showed that IgY and IgG could detect 0.1 g SCMV infected leaves using 1000‐times‐diluted antibodies. The IgY antibody was also confirmed to be reproducible and potentially applicable in plant disease diagnostics using an antibody‐based detection.

Protective effect of chicken egg yolk immunoglobulin (IgY) against Spiroplasma eriocheiris infection in Chinese mitten crab

The aquaculture of the Chinese mitten crab Eriocheir sinensis has become the leading and pillar of aquaculture in China. The tremor disease (TD) caused by Spiroplasma eriocheiris is the most destructive disease of E. sinensis. At present, the treatment of TD in crabs mainly depends on antibiotics. To protect the environment and the health of aquatic products, it is urgent to establish an effective and pollution-free green prevention and control technology. This study aimed to investigate the prevention and control of the TD by immunization with chicken yolk antibody (IgY). Anti-S. eriocheiris IgY was obtained by intramuscular injection of ultrasonically inactivated S. eriocheiris into chickens. After successful isolation and purification of IgY from eggs, ELISA and sensitivity tests were performed. Specific IgY was able to specifically react at a titer of 1:10,000 to antigen at a minimum concentration of 20 μg/mL and to inhibit the growth of S. eriocheiris in vitro. The affection of IgY in vivo to resist S. eriocheiris infection was demonstrated by feeding or injection of specific IgY. The copy number of S. eriocheiris in crab hemocytes with specific IgY administration was significantly less than in crabs that received normal IgY or without IgY. Meanwhile, the survival rate of E. sinensis with anti-S.eriocheiris- specific IgY using both injection and feeding methods significantly increased during S. eriocheiris infection. The electron microscope results showed that the structure of S. eriocheiris in specific IgY group was completely irregular and broken pin vivo. Finally, this study provides a reference for clarifying the potential role of specific IgY to help E. sinensis resist S. eriocheiris infection.

DNA starvation/stationary phase protection protein of Helicobacter pylori as a potential immunodominant antigen for infection detection.

Application of chicken egg yolk immunoglobulin Y (IgY) for Helicobacter pylori (H.pylori, HP) has gained much interest in recent years. Comparing with for treatment, IgY may be more advantageous when used for H.pylori detection. Nine strains of H.pylori with different genetic backgrounds were inactivated and used to immunize hens, respectively, for the preparation of polyclonal anti-H.pylori immunoglobulin Y (anti-HP IgY). The proteins of H.pylori with reactivity to anti-HP IgY were detected by Western Blot. The five protein bands that can be well recognized by anti-HP IgY of each group, and were prevalent in all nine strains were excised from SDS-PAGE gel, digested and identified by Nano-HPLC-MS/MS analysis. The potential of these identified proteins as antigen detection targets was then assessed by sequence analysis. Anti-HP IgY derived from each group of specific strain immunized hens can recognize self-strain and non-self-strain antigens well. Five immunodominant antigens were identified as chaperonin GroEL, flagellin A, urease subunit alpha, peroxiredoxin and DNA starvation/stationary phase protection protein. Sequences analysis showed that both peroxiredoxin and DNA starvation/stationary phase protection protein were present in all 1000 strains of H.pylori queried, and the amino acid sequences were highly conserved. The highest sequence consistency between the DNA starvation/stationary phase protection protein of H.pylori and non-Helicobacter organisms was 52.59%, and the consistent sites were scattered and there was no continuous long fragment consensus sequence. DNA starvation/stationary phase protection protein was identified as an immunodominant antigen of H.pylori and sequence analysis indicated that it could serve as a potential antigen target for the diagnosis of H.pylori infection.

Effect of anti-lipopolysaccharide of Escherichia coli antibody feeding for Holstein calves on ruminal lipopolysaccharide activity and plasma metabolites concentrations during pre- and post-weaning periods.

This study was performed to examine the effects of anti- lipopolysaccharide (LPS) of Escherichia coli chicken egg Yolk immunoglobulin (IgY) provided to calves for 7 weeks during the pre- and post-weaning periods on rumen LPS activity, plasma acute phase protein (APP) concentrations, and metabolic parameters. A total of 30 Holstein calves were randomly assigned to two groups of 15 each: an IgY group fed Anti-E. coli LPS IgY, and a control group fed whole egg powder as a placebo. The study was conducted on calves aged 3-10 weeks, weaned at 7 weeks. The ruminal LPS activity of the IgY group was approximately 60% lower than the control group at 10 weeks of age. Plasma APP and cytokine concentrations in the IgY group did not differ from those in the control group. The daily weight gain in the IgY group was significantly higher than the control group for the whole experimental period. Plasma albumin/globulin was lower (P<0.05), and plasma aspartate transferase concentration was higher (P<0.05) in the IgY group than in the control group during the experimental period. In conclusion, feeding Anti-E. coli LPS IgY for 7 weeks pre- and post-weaning remarkably reduced the rumen LPS activity and improved the daily weight gain. The impact of Anti-E. coli LPS IgY on LPS activities in the lower gastrointestinal tract, and elucidation as to the mechanism responsible for the improvement in daily weight gain require further investigation.

Immunoprotection effects of chicken egg yolk immunoglobulins (IgY) against Aeromonas veronii infection in Sinocyclocheilus grahami

Sinocyclocheilus grahami (S. grahami) has been artificially bred since it was domesticated in early 21st century. However, the large-scale breeding has exposed that S. grahami is susceptible to Aeromonas veronii (A. veronii). Herein, the specific immunoglobulin of yolk (IgY) against A. veronii was produced, which showed high specificity and sensitivity to bind with A. veronii. The stability scopes of temperature and pH were 20–60 °C and 4–7, respectively. The specific IgY significantly inhibited the growth of A. veronii by direct agglutination. Injection with the specific IgY protected S. grahami coping A. veronii infection by increasing ROS elimination speed and reducing the inflammatory damage, analyzed by activity of antioxidative enzymes (CAT, SOD and GSH-Px) and expression of TNF-α and IFN-γ genes, respectively. Our study will contribute to develop a type of antibiotic substitute IgY which could be applied to elevate the culture benefit in S. grahami.

Production and Evaluation of Chicken Egg Yolk Immunoglobulin (IgY) against Human and Simian Rotaviruses.

Producing specific antibodies in chickens is an attractive approach for diagnosis or therapeutic applications. Besides the high immunoglobulin Y (IgY) yield transferred to the egg yolk and its suitability for large-scale production, such an approach is more bioethical for animal maintenance. The IgY technology offers new possibilities for application in human and veterinary diagnostics and therapeutics, including strategies for treating severe intestinal diseases in children, particularly in emerging countries. Herein, we describe the production and purification of polyclonal antibodies against rotavirus group A (RVA) in immunised hens aiming at its application in prophylaxis and treatment of rotavirus-induced diarrhoea. For this purpose, we inoculated Rhodia laying chickens (Gallus gallus domesticus) with two or three doses of RVA combined with adjuvants or only adjuvants (control group). As the egg-laying period began, the yolk protein purification processes yielded a high concentration of specific IgY, the highest titre resulting from the group of hens that received three doses of the immunogen. The purified IgY blocked the functional activity of RVA in MA-104 cells, thus confirming the neutralisation ability. Therefore, anti-RVA IgY could be a promising candidate for pre- and post-exposure prevention or treatment of rotavirus-induced diarrhoea.

Potential for Neutralizing Chicken Egg Yolk (Igy) Generated against Mastitis Bovine, Caused by Pseudomonas aeruginosa and Klebsiella pneumoniae

Introduction: Mastitis is a complex disease, defined as an inflammation of parenchymal cells of mammary glands. It is a most common disease in dairy cattle. More than 250 microbes are found to cause mastitis. Recent report shows that total loss of about $2295/month in worldwide due to mastitis.&#x0D; Researchgap/Challenges: Chicken egg yolk immunoglobulin (IgY) provides an inexpensive and effective source of antibodies for the passive immunization of animals. It is a promising alternative for the treatment and prevention of bacterial infections, and has shown to be effective against a number of pathogens.&#x0D; Aim: To develop an inexpensive and effective source of antibodies by using chicken egg yolk against mastitis caused by Klebsiella pneumoniae and Pseudomonas aeruginosa in bovine.&#x0D; Methodology: Bovine mastitis infected milk sample was collected in and around Coimbatore, India. Initial step was to isolate and identify the microbes like Klebsiella pneumoniae and Pseudomonas aeruginosa from milk samples of cattle infected with mastitis. Then to prepare and standardize the whole cell antigen of Klebsiella pneumonia and Pseudomonas aeruginosa followed by generation of antibodies against the prepared antigens in 21-weeks old white Leghorn chickens. Next step includes purification and characterization of anti-Klebsiella pneumoniae IgY and anti - Pseudomonas aeruginosa IgY antibodies from the egg yolk of immunized chickens. Finally, to evaluate the specificity of the purified IgY by Indirect ELISA and to measure the inhibitory activity of the IgY against the pathogens by growth inhibition assay.&#x0D; Results: Cell antigen was prepared. Anti- Klebsiella pneumoniae IgY and anti - Pseudomonas aeruginosa IgY were recovered from chicken egg yolk that was detected by protein estimation (Lowry et al technique) and ELISA. In Lowry et al method (1951) total anti P. aeruginosa IgY was found to be 0.511 mg ml-1 and total anti K. pneumoniae IgY was found to be 0.522 mg ml-1.&#x0D; Conclusion: Generated antibodies were identified as very specific to the immunization antigen. Based on invitro studies generated antibodies were specific to immunized antigen. It concluded that it will be useful or therapeutic after further conformation.

Protective Effects of Chicken Egg Yolk Immunoglobulins (IgYs) against Vibrio vulnificus Infections.

Vibrio (V.) vulnificus infection is a rare disease whose death rates exceed 50% despite aggressive antibiotic treatment and surgical debridement. The aim of this study was to assess the ability of specific anti-V. vulnificus immunoglobulins Y (IgYs) for preventing and treating V. vulnificus infections. IgYs were produced by immunizing egg laying hens with inactivated whole cell bacteria. Peritoneal cytokines, blood's bacterial load, and survival curves were obtained from both prophylactic and therapeutic mouse models. The results showed that the specific IgYs (i) inhibited the growth of V. vulnificus in vitro, (ii) dramatically reduced the inflammatory response and blood's bacterial load, and (iii) improved the survival rate of V. vulnificus-infected mice. These results prove that anti-V. vulnificus IgYs can be markedly effective means for the prophylaxis and the therapy of V. vulnificus infections.

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.

Passive protection of Japanese pufferfish (Takifugu rubripes) against Vibrio harveyi infection using chicken egg yolk immunoglobulins (IgY)

In recent years, Vibrio harveyi has become a significant cause of skin ulceration of Japanese pufferfish (Takifugu rubripes), and the outbreaks have been responsible for mass mortalities and high economic losses. Thus, the main purpose of this study was to evaluate the protective effect of chicken egg yolk immunoglobulins (IgY) against V. harveyi infection in the T. rubripes. In the present study, hyperimmune anti-V. harveyi IgY was extracted from eggs laid by Hy-Line Brown hens immunized with formaldehyde-inactivated whole V. harveyi cells. The purity of the IgY after separation from egg yolk was approximately 82.4% as assessed by SDS-PAGE. The maximum IgY titer (ELISA) in water-soluble fractions was up to 1:130000 at week 12th of immunization of hens. In-vitro experiment revealed that the IgY inhibited the growth of V. harveyi in liquid medium by a concentration-dependent manner in the range of 1 to 10 mg/mL and on agar plates (P < 0.05). The effect of 10 mg/mL of IgY was similar to the 0.1 mg/mL of streptomycin within 7 h (P > 0.05). Confocal laser scanning microscope (CLSM) assays revealed that anti-V. harveyi IgY caused bacterial agglutination. The T. rubripes intramuscularly injected with 100 μL of V. harveyi (5.4 × 107 CFU/mL) and the IgY (10 mg/mL) showed a survival rate of 80% (P < 0.001). Dietary supplementation of 10% egg yolk powder significantly enhanced the resistance to V. harveyi infection with a survival rate of 50.0% (P < 0.001), as against 100% death in the positive control group. Immersing in 0.5 g/L aqueous IgY resulted in a 60.0% survival in challenged fish (P < 0.001), along with increase in the levels of non-specific immune-related enzymes (SOD, POD, NOS, ACP and LYZ) (p < 0.05). While 1 mg/mL of IgY significantly enhanced the phagocytosis of V. harveyi by macrophages (p < 0.05). These results revealed that IgY has great prospects in protecting T. rubripes against V. harveyi infection.

Protective effect of chicken egg yolk immunoglobulins (IgY) against enterotoxigenic Escherichia coli K88 adhesion in weaned piglets

BackgroundEnterotoxigenic Escherichia coli K88 (E. coli K88) are considered as a major cause of diarrhea and death in newly weaned piglets. Oral passive immunization with chicken egg yolk immunoglobulins (IgY) have attracted considerable attention for treatment of gastrointestinal infection due to its high specificity. In this study it was estimated the protective effect of anti-K88 fimbriae IgY against E. coli K88 adhesion to piglet intestinal mucus in vitro and to investigate the potential use of IgY for controlling E. coli-induced diarrhea in weaned piglets in vivo.ResultsE. coli K88 was incubated with IgY for 24 h, and the bacterial growth profiles showed that specific IgY with a concentration higher than 5 mg/mL was observed to significantly inhibit the growth of E. coli K88 compared to nonspecific yolk powder in a liquid medium. Moreover, pretreatment with 50 mg/mL of IgY was found to significantly decrease the adhesion ability of E. coli K88 to porcine jejunal and ileal mucus, further supported by the observations from our immunofluorescence microscopic analysis. In vivo, administration of IgY successfully protected piglets from diarrhea caused by E. coli K88 challenge. Additionally, IgY treatment efficiently alleviated E. coli-induced intestinal inflammation in piglets as the gene expression levels of inflammatory cytokines TNF-α, IL-22, IL-6 and IL-1β in IgY-treated piglets remained unchanged after E. coli K88 infection. Furthermore, IgY significantly prevented E. coli K88 adhering to the jejunal and ileal mucosa of piglets with E. coli infection and significantly decreased E. coli and enterotoxin expression in colonic contents.ConclusionOutcome of the study demonstrated that IgY against the fimbrial antigen K88 was able to significantly inhibit the growth of E. coli K88, block the binding of E. coli to small intestinal mucus, and protect piglets from E. coli-induced diarrhea. These results indicate that passive immunization with IgY may be useful to prevent bacterial colonization and to control enteric diseases due to E. coli infection. The study has great clinical implication to provide alternative therapy to antibiotics in E coli induced diarrhea.

Effect of chicken egg yolk immunoglobulins on serum biochemical profiles and intestinal bacterial populations in early-weaned piglets.

This study was conducted to test the hypothesis that dietary supplementation with anti‐E. coli, chicken egg yolk immunoglobulins (IgY), may affect early weaned piglet (EWP) intestinal functions and enteric micro‐organisms. One hundred and forty‐eight ([Landrace × Yorkshire] × Duroc) piglets, weaned at age day 21, were randomly assigned to receive one of three diets for 14 days. Treatment group one (control group) was fed the base diet. Treatment group two (antibiotics group) was fed the base diet which was supplemented with 100 ppm colistin sulphate and 15 ppm enramycin; treatment group three (IgY group) was fed the base diet which was supplemented with 500 mg/kg anti‐E. coli IgY. The study evaluated the effects on EWPs of IgY on growth, serum biochemical, inflammatory profiles and also digestion content intestinal bacterial populations. Results showed no significant difference in diarrhoea rates between IgY‐fed EWPs and antibiotic‐treated EWPs. Serum biochemical analysis showed that EWPs fed an IgY‐containing diet had both lower (p < 0.05) cholesterol and low‐density lipoprotein compared to antibiotic‐treated EWPs. Escherichia coli populations measured in IgY‐fed EWP ileal contents, compared to the control group, were significantly reduced (p < 0.05). Enterococcus, Lactobacillus, Clostridium and Bifidobacterium populations were unaffected by the IgY treatment. Larger (p < 0.05) Enterococcus populations and lower (p < 0.05) expression levels of heat‐stable enterotoxin b (STb) were observed in IgY‐fed EWP caecal digesta compared to the control group. Enteric Lactobacillus significantly decreased (p < 0.05) in EWPs fed antibiotics while it was unaffected by IgY treatment. Dietary supplementation with anti‐E. coli IgY has the potential to suppress enteric E. coli growth, but not Lactobacillus, Clostridium and Bifidobacterium. This promotes and maintains a healthy EWP intestinal environment. These findings suggest that IgY may be used as an alternative to antibiotics in EWP diets.

Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Enterovirus type 71-immunized chicken egg yolk immunoglobulin has cross antiviral activity against coxsackievirus A16 in vitro.

To exploit a cross passive immunotherapy for enterovirus-induced hand-foot-and-mouth disease (HFMD), the cross antiviral activity of a neutralizing antibody against enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) was investigated in vitro. White Leghorn specific-pathogen-free chickens were immunized with EV71 antigens and a specific isolated immunoglobulin (IgY) was prepared from the chicken egg yolk. IgY was further purified and characterized by SDS-PAGE, ELISA, western blotting and bidirectional immune agar diffusion testing. The antiviral activity and dose-response of the IgY were determined by assessing the cytopathic effect in rhabdomyosarcoma (RD) cells in vitro. It was indicated that the levels of IgY were increased at day 7, peaked at week 7 and were maintained at a higher level for 4 weeks following immunization when compared with the negative control. The results of western blotting and bidirectional immune agar diffusion testing revealed that the IgY had cross-binding properties in EV71 and CVA16 strains through targeting the envelope proteins (VP0, VP1 and VP3) of EV71 and CVA16. Neutralization assay results indicated that the infectivity of EV71 and CVA16 strains in RD cells was cross-blocked by IgY in a dose-dependent manner. To conclude, these findings indicate that IgY has cross antiviral activity against EV71 and CVA16 in vitro, and could potentially be developed as a passive immunotherapy for EV71- and CVA16-induced HFMD.

Preparation and identification of chicken egg yolk immunoglobulins against human enterovirus 71 for diagnosis of hand-foot-and-mouth disease

Human enterovirus 71 (EV71) is one of the major pathogens that causes hand-foot-and mouth disease, and there is an urgent need for rapid diagnosis of EV71 virus infection for early antiviral treatment. The aim of this study was to prepare chicken egg yolk immunoglobulin Y (IgY) for the diagnosis of enterovirus type 71 infection. The antibodies were raised by intramuscular immunization of laying hens with inactivated human EV71 and isolated from the egg yolk by multiple steps of polyethylene glycol 6000 extraction. The average concentration of IgY antibody was 26.60 mg/mL during the whole immunization. After the first immunization, the IgY titer gradually increased, and reached the peak on the 55th days. Meanwhile, the use of western blotting test demonstrated that specific IgY binds specifically to capsid proteins VP2 and VP3 of EV71 virus. Furthermore, a facile one-step method based on turn-on fluorescence sensing was developed by using IgY antibodies, which can detect EV71 virus at low concentrations of 104 PFU/mL and was 94.44% coincidence with RT-PCR in 30 clinical samples. These findings indicate that EV71-IgY antibodies are an easily prepared and rich source of antibodies that offers a potential alternative strategy for routine screening of EV71 infection.

Enterobacter cloacae associated with mass mortality in zoea of giant freshwater prawns Macrobrachium rosenbergii and control with specific chicken egg yolk immunoglobulins (IgY)

Outbreaks of mass mortality among cultured zoea of M. rosenbergii occurred in different hatcheries of Gaoyou country of China in April 2017. Of the isolates obtained from the diseased zoea, XL3–1 was pathogenic to the zoea and postlarva of M. rosenbergii in the virulence test, with a LD50 value 3.2 × 106 CFU/mL and 3.5 × 106 CFU/mL, respectively. XL3–1 was identified as Enterobacter cloacae by biochemical characteristics and molecular tools. Detection of virulence-associated genes by PCR indicated that XL3–1 was positive for irp2, fhuA, sodB, sltA, flaD and OmpX. According to the antibiotic resistant assay, the isolate was sensitive to quinolones, while they were resistant to cephalosporins and penicillins. This study also investigated the protection of specific chicken egg yolk antibodies (IgY) in E. cloacae-infected M. rosenbergii. The highest titer against E. cloacae of the obtained specific IgY by ELISA was 1: 213. M. rosenbergii treated with specific egg yolk powder could achieve the survival rate of 67% after E. cloacae infection, while M. rosenbergii treated with non-specific egg yolk powder or no egg yolk powder could achieve survival rates of 20% and 17%, respectively. As well, the bacterial burden in hepatopancreas was significantly lower in M. rosenbergii treated with specific egg yolk powder than those treated with non-specific egg yolk powder. This study clarified the virulence characteristics and antibiotic resistance of E. cloacae, and provided a strategy to control the disease by using specific IgY.

Features of chicken egg yolk immunoglobulin (IgY) against the infection of red-spotted grouper nervous necrosis virus

Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most important viruses which mainly infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide. Currently, there are no effective vaccines available for the fish larva infected with NNV. Immunoglobulin yolk (IgY) origin of oviparous animals is passed from the blood serum and concentrated in the egg yolk. With the advantages of high yield, cost-effectiveness, and high stability, IgY can be widely used in passive immunization, especially in young animals in which adaptive immunity is not fully developed. In this study, we have cloned and expressed the recombinant capsid protein of RGNNV in Escherichia coli and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens. Water-soluble fractions (WSF) of the specific IgY were isolated from egg yolk and purified by two-step precipitation with saturated ammonium sulfate salting. By Enzyme linked immunosorbent assay (ELISA), the titer of the IgY reached a peak at the 6th week post of immunization and had a strong stability at a wide range of temperature, pH, and pepsin enzyme digestion. The purified IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line (GF-1), indicating that it was highly specific and effectively recognized RGNNV. The results will pave a new way for the prevention of RGNNV infection.

Protective effects of chicken egg yolk immunoglobulins (IgY) against experimental Aeromonas hydrophila infection in blunt snout bream (Megalobrama amblycephala)

The emergence of multi antibiotic resistance by the pathogens and toxic impacts on host metabolism has opened new perspectives to rational novel vaccine techniques. Outbreaks of Aeromonas hydrophila in aquaculture caused high mortality throughout the world and resulted in the extensive economic loss in the aquaculture industry. In this study, we report the efficacy of anti-A. hydrophila IgY antibodies by passive vaccination and its prophylactic or therapeutic effects against A. hydrophila in blunt snout bream. Inactivated A. hydrophila immunized hens produced effective IgY antibodies that were stable at temperatures less than 60 °C or the pH value was >4. The specific IgY can be bound directly to A. hydrophila that efficiently agglutinated and inhibited the bacterial growth in a dose-dependent manner. The specific IgY had significantly enhanced the phagocytosis activity of macrophages and resulted in rapid bacterial clearance. Anti-A. hydrophila IgY antibodies significantly increased macrophage mediated respiratory burst, including nitric oxide and superoxide anion production and subsequently killed the pathogen. Histopathological studies of intestine and spleen from vaccinated blunt-snout bream challenged with A. hydrophila showed the structural integrity of the organs was maintained intact from the bacterial injury. In addition, the prophylactic and therapeutic immunization, protected the blunt snout bream and the survival is approximately about 60% and 50%, respectively. These data suggest that specific IgY has the potential for protecting blunt snout bream against A. hydrophila infection and show promise for the future development of harmless vaccines.

Purification of chicken IgY by binding capture using elastin-like polypeptide-tagged immunoglobulin-binding domain of streptococcal protein G

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin, but its practical application is limited due to the complex purification procedure. In this study, the C2 domain of streptococcal protein G (SPG) with the binding affinity for chicken IgY was expressed in E. coli as an elastin-like polypeptide (ELP) fusion protein, and purified to a high purity by inverse transition cycling (ITC). Binding experiments showed that chicken IgY could bind to and eluted off the ELP-C2 fusion protein in pH-, temperature- and/or time-dependent manners. By using the ELP-C2 protein, a simple chicken IgY purification method was developed, and its purification performance was compared with that of ammonium sulfate precipitation and ethanol fractionation. Quantitative SDS-PAGE analysis showed that the ELP-C2 binding capture method provided a chicken IgY purity of 96.3% and a recovery of 64%, both of which were significantly higher than that of the two traditional methods The ELP-C2 binding capture method could be accomplished within 3h, in contrast to 30.3h for ammonium sulfate precipitation or 4.3h for ethanol fractionation. These data suggest that the ELP-C2 binding capture was a simple, efficient and cost-effective method for purification of chicken IgY.