Production And Features Of Egg Yolk IgY Against Red-spotted Grouper Nervous Necrosis Virus And Aeromonas Hydrophila

Nervous necrosis virus（NNV）,the major infectious agent which causes viral nervous necrosis（VNN）,infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide.Currently,there are no effective antiviral measures or vaccines available for the fish larva infected with NNV.Aeromonas hydrophila is the major pathogen causing fish bacterial sepsis which is an acute infectious disease causing serious loss of fresh water fish culture in China.Due to the abuse of antibiotics for the prevention of A.hydrophila inection,resulting in outbreaking of drug resistant bacteria,the new prevention and treatment measure for the A.hydrophila inection is urgently needed to be developed.Immunoglobulin of 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 which adaptive immunity is not fully developed.In this study,we cloned and expressed the recombinant capsid protein of red-spotted grouper nervous necrosis virus（RGNNV）and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens.While,inactivated A.hydrophila immunized hens produced specific anti-A.hydrophila IgY.The stability and the immune protection of the IgYs were explored.The major results are as follows:1.The preparation and the titer of the IgYs.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 obtained anti-RGNNV IgY reached a peak level at the 6^th week post of immunization,while the peak was at 7^thh week for anti-A.hydrophila IgY.The highest titer of anti-RGNNV IgY and anti-A.hydrophila IgY was 1:51200 and 1:25600,respectively.Both of them lasted at high titer for some weeks.2.The physic-chemical features of the IgYs.The stability of anti-RGNNV IgY showed excellent stability at a broad range of temperature（below 70℃）,pH value（2 to 10）and could remain steadily active in pepsin at pH of 3-10.Anti-A.hydrophila IgY was stable at temperatures less than 60℃or the pH value was>4.Replication of RGNNV was blocked when anti-RGNNV IgY was heated at 30-60℃or treated with various pH（2-10）or digested by pepsin at pH of 3-10.Altogether,specific IgY showed a strong stability in a wide range of temperatures,pH and pepsin enzyme.3.The immune protection of anti-RGNNV IgY.Western blot analysis showed that the anti-RGNNV IgY had highly specificity and affinity which could recognize RGNNV capsid protein.The anti-RGNNV IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line（GF-1）,indicating that anti-RGNNV IgY was effection in prevention of the infection of RGNNV in cells.4.The immune protection of anti-A.hydrophila IgY.24 hours before the challenge with A.hydrophila,blunt-snout bream（Megalobrama amblycephala）was injected with anti-A.hydrophila IgY or non-specific IgY or PBS,respectively.The results showed that the survival rate one week after the challenge was 60%for anti-A.hydrophila IgY group,10%for non-specific IgY or PBS group.While,12 hours after the challenge with A.hydrophila,blunt-snout bream（Megalobrama amblycephala）was injected with anti-A.hydrophila IgY or non-specific IgY or PBS,respectively.The results showed that the survival rate one week after the challenge was 50%for anti-A.hydrophila IgY group,100%for non-specific IgY or PBS group.The immune protection results were further confirmed by histopathological studies,indicating that anti-A.hydrophila IgY was effective in prevetion and therapy of A.hydrophila infection.