Preparation And Efficacy Evaluation Of Egg Yolk Immunoglobulin (IgY) Against Sea Cucumber (Apostichopus Japonicus) Skin Ulceration Syndrome Caused By Vibrio Alginolyticus

Apostichopus japonicus skin ulceration syndrome has become the most serious disease for its strong infectivity and high mortality.The actual production mainly relies on antibiotics and chemical drugs to control,but in the long run,bacterial resistance will become more and more serious.At the same time,many problems such as drug residues,ecological environment imbalance and the impact on human health have attracted the attention of scholars at home and abroad.It is urgent to develop green,safe and efficient antibiotics substitutes.In order to explore the death causes of sea cucumber（Apostichopus japonicus）with skin ulceration syndrome from a local farm in Dalian,a dominant bacterial strain AP-1 was isolated from the diseased sea cucumbers.The morphological observation,16S rRNA gene sequence,physiological and biochemical characteristics results showed that it was Vibrio alginolyticus.The strain was artificially infected back to the healthy sea cucumber by the means of immersion infection,body wall wound immersion infection and intraperitoneal injection infection respectively.The results showed that the immersion infection did not show pathological symptoms,whereas other two methods showed different degrees of dose-dependent lethality.The LC₅₀of body wall wound immersion infection and LD₅₀ of intraperitoneal injection infection with the isolate were 5.23×10⁶ CFU/mL and 3.08×10⁴CFU/g,respectively.Virulence gene detection showed that the strain could be positively amplified with the seven virulence genes including toxR,FlaA,Collagenase,fur,AspA,ompW and tlh.The results of drug sensitivity showed that the strain was resistant to six antibiotics,ampicillin,piperacillin,ogmentine,cefazolin,cefotaxime and amikacin,and was intermediate or sensitive to other tested antibiotics.The Egg yolk immunoglobulin was prepared with inactivated Vibrio alginolyticus as antigen and purified by water dilution,freeze thawing,salting,ultrafiltration concentration method.The purity of IgY was 90.13%.Enzyme linked immunosorbent assay showed that the highest titer of the antibody was 1:64000,which lasted for 2 weeks.The total IgY content in the water soluble fraction was about 1.6 mg/mL,and the highest specific IgY content was0.256±0.032 mg/mL,accounting for 16.06%of the total IgY.The results of immunofluorescence electron microscopy and transmission electron microscopy showed that IgY can specifically bind to Vibrio alginolyticus AP-1 and agglutinate the bacteria.In vitro antibacterial growth experiments found that specific IgY can significantly inhibit the growth of bacteria.Bacterial activity testing experiments showed that the activity was reduced,and the internal structures of pathogenic bacteria showed that the surface of the bacteria was blurred and the degree of cytoplasmic vacuolation was increased.Crystal violet staining and microbial adhesion to hydrocarbons experiments showed that specific IgY can reduce the formation of Vibrio alginolyticus biofilm and enhance the hydrophobicity of the bacterial surface.In animal experiments,the survival rate of infected sea cucumbers was significantly improved by IgY injection after intraperitoneal injection infection and body wall wound immersion infection after immersing with IgY,indicating that specific IgY can play a good protective effect on sea cucumbers.Enzyme linked immunosorbent assay showed that after healthy sea cucumbers were immersed in IgY,the coelomic fluid still had IgY activity after24 h;immunofluorescence experiments showed that IgY could be distributed on the respiratory trees,and speculated that the respiratory tree was one of the routes for IgY to enter coelomic fluid and other tissues from seawater.In conclusion,the egg yolk immunoglobulin prepared by using the isolated Vibrio alginolyticus AP-1 as antigen has a good protective effect on sea cucumbers,which can provide a scientific basis and reference for the further study of the antibacterial mechanism of IgY and the prevention and control of sea cucumbers diseases.