Preparation And Application Of Anti-Spiroplasma Eriocheiris Immunoglobulin Yolk(IgY) In Chinese Mitten Crab,Eriocheir Sinensis

Chinese mitten crab,Eriocheir sinensis,is one of important species in aquaculture in China.Tremor Disease(TD),caused by Spiroplasma eriocheiris infection,is the most damaging disease of this crab.At present,the treatment of this disease mainly relies on antibiotics,and the abuse of antibiotics and other chemical drugs may lead to a series of problems,such as drug residues,the emergence of drug-resistant bacteria,environmental pollution and aquatic animal immune system damage.Therefore,in order 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.The purpose of this study was to explore a method of immunization using Ig Y to prevent and control the disease.The study method was conducted with three main steps: 1.Ig Y isolation and purification;2.ELISA test and growth inhibitory test;3.Ig Y neutralization experiment and application.Chickens were immunized with ultrasonically crushed S.eriocheiris,as the antigen.Three injections were made with an interval of 2 weeks between each injection.After eggs were harvested,Ig Y was then isolated and purified.Then the SDS-PAGE method was used,to verify the successful of the immunization.After IgY successfully isolated,indirect ELISA test were conducted to determine the Ig Y titer.Afterwards,growth inhibitory test were also conducted to determine the proportion of use.The best candidate as specific Ig Y was obtained from chickens injected with 400 μg/m L antigen with Ig Y titer 1:10,000;at week 4 after the first injection;and at the Ig Y usage ratio of 1: 4.Subsequently,for IgY neutralization experiment and application,crabs were challenged by S.eriocheiris and then fed with Ig Y feed.At the same time,other different crabs were challenged by Ig Y and S.eriocheiris mixed injection.The results of in vivo experiments showed that crabs that received specific Ig Y and were attacked by S.eriocheiris survived significantly more than crabs that received normal(non-specific)Ig Y or without Ig Y.The results of in vitro experiments showed that the copy amount of S.eriocheiris DNA in crab blood cells with specific Ig Y administration was significantly less than crabs that received normal Ig Y or without Ig Y.In addition,the electron microscope observation of crabs hemocyte shown that the structure of S.eriocheiris with specific Ig Y changed different from the typical spiral structure of spiroplasma,shown completely irregular and broken phenomenon.Finally,this study is expected to provide a reference for the potential effect of specific Ig Y against S.eriocheiris attack,and could be practiced in aquaculture.