Study On The Preparation Of Cryptosporidium Baileyi IgY And Its Application

Egg yolk immunoglobulin,also called IgY,is one of three principal classes immunoglobulins in avian sera.Because the chicken isn't the mammals,and has relatively remote phylogenetic relationship with human,the pathogens of human and the proteins which is highly conserved of the mammals can produce the immune response activity strongly.Another,the thermal and acid stability of IgY are good,preserving 5 years in 4℃or placing 6 months at room temperature,the activity of antibody maintained good in the conjugate.The specificity of IgY coming from chickens is easy to select material,to extract,and can large scale production,so has large application foreground in passive immunization treatment of food and oroal fields.The Cryptosporidiosis is the causive agent of cryptosporidium in a range of animal species and human beings,the major clinical manifestations were diarrhoea.It distributes worldwide,and has been listed one of the most common 6 diarrhoeas.Though recently the research on the prevention and treatment of Cryptosporidiosis cause more attention of the experts in our country and overseas,they do not select a efficient drug to prevent and treat it.It is necessary to study further.The aim of article is preparation of anti- cryptosporidium specific of yolk antibody,the protective mechanism of yolk antibody was also discussed.Using of cryptosporidium oocysts preserving in lab,we take the improving Sheather sugar gradients centrifugal method purified oocysts,after low-speed centrifugal combined with filter membrane micro-removing bacteria,oocysts antigen through freezed/thawed and ultrasonic treatment,with completely/non-complete freund's adjuvants to preparation of immune antigen,the healthy chickens are immuned 6 times by antigen increaing's gradients immuning method;The yolk antibody were separated and purified by acidizing water dilution -salt evolution method,and then comparativing with all results.The indirect ELISA method is established to detect antibody titer, deter.min-lation of purity by SDS-PAGE,deter.min-lation of concentration of protein by ultraviolet spectrophotometry.The results shows:water dilution two steps-ammonium sulphate salt evolution method has good results,concentration of protein is 9.44mg/ml,an egg could provide the minimum 94.4mg antibody.The obtained yolk antibody was identidied habing cryptosporidium specifics by indrect ELISA.The result of SDS-PAGE shows IgY and serum's IgY have homology that it transfer from serum to yolk.The stability of IgY was meassured by indrect ELISA,results show:①IgY was meassured titer after putting into 25℃-80℃water bath 15 mins;②incubated for 2 hours with 37℃incubator,regulating different pH 7.2～7.4 with NaOH and HCL,meassuring sample titler.The activity of IgY don't change obviously in pH 4～10,and has no effect at 65℃.Results show IgY has good stability.,and provid a reliable basis for the clinical applicationTaking specific IgY obtained from this studying to treat cryptosporidium,the test divided into 7 groups:blank control group,infection group,negative IgY treatment group,Cryptosporidium baileyi IgY treatment group,Cryptosporidium andersoni IgY treatment group of high concentration,middle concentration,low concentration,infecting C.baileyi oocysts 3.2×10~6 every chicken after feeding antibody 2 days.Observing every groups as follows:oocysts' changing,clinical symptoms,bursal index,effecting to increaing weight.Results show:there aren't obvious difference between negative IgY treatment group and infection group in every index;compareing negative IgY treatment group and Cryptosporidium andersoni IgY treatment group of three doses,three doses groups are discharging oocysts peak in 10 days,and its quantity are lower than negative IgY treatment group,especially high concentration in the lowest;about during of oocysts:Cryptosporidium andersoni IgY treatment group of low concentration begains to discharge oocysts after infected 3 days,and its quatity is low 12%than negative IgY treatment group continuity is 19 days,less 1 day than negative IgY treatment group;middle concentration dose's index are lower than 28%,during 18 days,less 2 days,respectively;high concentration dose's index are lower than 43%,during 18 days,less 2 days,respectively;all those suggest that using Cryptosporidium andersoni IgY could prevent and treat oocysts discharging amounts and continuity decreasing.It reflect Cryptosporidium andersoni IgY inhibits oocysts from another side with a dosage-related.Comparing between Cryptosporidium baileyi IgY treatment group and Cryptosporidium andersoni IgY treatment group in different three doses:the quantity of oocysts is lower 45%than three treatment groups,duration are 18 days.This suggests that cryptosporidium baileyi IgY could treat oocysts infecting and decrease OPG.This trial has successed in preparing high activity anti-cryptosporidium specific IgY,it has obvious effect in increasing immune of chicken and preventing specific cryptosporidium infecting, and Cryptosporidium andersoni IgY has cross-protection to coxiella cryptosporidium.