| Riemerella anatipestiffer(RA)is the pathogen of infectious serositis in ducks,geese and other poultry,and it is gram-coccobacilli,with at least 21 serotypes and lack of cross-immune protection.In China,infectious serositis is still one of the main common diseases that endanger goose and duck breeding industry,caused huge economic losses.There are many reports on inactivated vaccine and live vaccine of this disease,and inactivated vaccine of serotype 1,serotype 1 and 2 bivalent oil emulsion has been commercialized in China.However,the practical application of these vaccines hasn't effectived.The analysis showns that the immune induction period of RA inactivated oil emulsion vaccine is 10-14 days,which is the immune blank period.For farms that have not established good biosafety measures,ducks and geese are infected with RA during the immune blank period,and these results has immune failured.Our research group has proved that chicken anti-RA egg yolk antibody(C-RA Ig Y)has a good passive immune protection effect on ducks and geese,and can made up for the immune blank period with RA oil emulsion inactivated vaccine.The antibody titer of C-RA Ig Y products determined by Agar gel immunodiffussion test(AGID)of RA extracted by hot phenol antigen(RA-PEAg)is related to the efficacy of passive immune challenge test of susceptible ducks.RA sterile filtrate has immunoprotective effect.In order to compare the difference of immune efficacy between RA bacteria and sterile filtrate,this study carried out the research on RA exopolysaccharide antigen and its production rule,and obtained the following results.1.LPS antigen ectracted by RA.The RA soluble antigens extracted by Ult RAsonic treatmented(US),High pressure cracked(HP)and Hot phenol extracted(PE)were compared,and the AGID was detected by chicken anti-RA specific egg yolk antibody(RA-CIg Y,whose immune protection effect was proved by duck passive immune protection test).The results showed that the precipitation lines of RA antigens prepared by three methods,namely HPAg,US Ag and PEAg,fused with each other,and the precipitation lines of serotypes 1 and 2 crossed.PEAg has high sensitivity,which is 1-2 titers higher than US Ag and HP Ag.After PEAg were treated with 10% Trichloroacetic acid(TCA),the protein content of two batches of RA serotype 1 SC strain decreased from 106.38?g/m L and126.38?g/m L to 41.86?g/m L and 60.19?g/m L,and the protein removed rates were60.65% and 52.37%.PE Ag protein content of two batches of RA serotype 2 AF strain decreased from 95.43?g/m L and 47.10?g/m L to 30.43?g/m L and 6.38?g/m L,and the protein removed rates were 68.13% and 86.45%.According to AGID test results,PET Ag precipitation lines are relatively central and clear,and the precipitation line of antibody high dilution hole is especially close to peripheral antibody hole,which leads to a slight decrease of sensitivity by 0.5 titer.PE Ag were treated with chloroform(Sevag method,chloroform: n-butanol,PES Ag).The protein content of two batches of RA serotype 1 SC strain was 70.90?g/m L and 50.19?g/m L;The removal rates of protein were 33.36% and 60.29%,the protein contents of two batches of serotype 2AF strain were 26.14?g/m L and 1.38?g/m L,and the removal rates of protein were72.61% and 97.07%.After 30 days of cold storage,PET Ag were not affected and PES Ag were stored for 14 days,and 2 batches of 3 samples of serotype 2 AF strain failed.The PE Ag and after deproteinized PET Ag or PES Ag,limulus amebocyte lysate(0.25EU/m L)were qualitatively positive.The result showed that PE Ag were LPS Ag with good specificity and stability.2.The AGID and indirect ELISA test were established for detection of RA-PET Ag concentration.The RA-CIg Y samples with AGID titers of 0,2 and 5log2 were used as reference antibodies,and The AGID and indirect ELISA test were established for detection of RA-PET Ag concentration.The AGID results showed that RA-CIg Y samples(AGID titer were 5log2)were 4-fold dilution and then added to the central hole,and RA-PET Ag or Filtrated solution(FS)samples were serial 2-fold dilution and added to the peripheral hole.the highest dilution of AGID with precipitation lines were determined as RA-PET Ag or Extracellular polyscharride antigen(EPS)concentration.The Samples with PET Ag concentration of 1:64 were used for indirect ELISA antigen.RASC-PETAg and RAAF-PETAg were diluted 10-fold or 50-fold respectively and coated with ELISA plate.HRP-goat anti-chicken Ig Y was used to establish indirect ELISA for detected antibody titer of RA-CIg Y samples.30 negative samples were diluted 200-fold,and the average A450 values were 0.128 and0.126.Three samples were selected as negative reference antibodies(Cn).With2.1x Cn A450 as the critical value.Specificity test was carried out with Ig Y samples of chicken anti-AIV of type A H9 subtype and egg yolk antibody samples of chicken anti-duck hepatitis virus.The results showed that both samples were negative at 1:400dilution.The titer of CIg Y samples of RA serotype 1 SC strain and RA serotype 2 AF strain reached 1:25600 and 1:6400,and Cross-detection between different serotypes was negative.The results showed that indirect ELISA was highly sensitive and specific for detecting RA LPS antigen.3.Extraction of extracellular antigen of RA.RA was cultured to 16 hours by TSB,and the thallus precipitates were discarded by centrifugation twice.The supernatant was sterilized and filtered by 0.22?m filter to prepare FS samples.Then Cetyltrimethylammonium Bromide(CTAB)was used to obtain the sample(FSE).The results showed that FSE prepared at CTAB concentration of 0.1%?0.8% and 4??8?reacted with RA-CIg Y to form aprecipitation lines,which merged with the precipitation lines of RA-PE Ag.The concentration of CTAB was the highest in 0.2%group.FSE samples of RA serotype 1 SC strain(the concentration measured by AGID was 1:4)were injected intravenously and intramuscularly into ducklings aged 7 and14 days.Within 48 hours,the temperature of experimental ducks changed less than0.5?,and there was no abnormality in spirit,walked,feeded and feces.The results showed that there were polysaccharide antigen with the same reactivity as LPS antigen in the sterile filtrate of RA culture,that was extracellular polysaccharide antigen.4.The generation rule of RA-EPS Ag.The RA-FSE samples were prepared by TSB and LB culture respectively.after serial dilution,were mixed with reference RA-CIg Y antibody 1:200 dilution,after combine at 37? for 1hours,the supernatant was centrifuged for indirect ELISA detection.With RA-PET Ag as reference and1:400 dilution of RA-CIg Y as control,the A450 reduction value of blocking tube samples were used to calculated the blocking rate.The results showed that the blocking rate of serotype 1 SC strain were 58.80% in TSB group and 44.64% in LB group,and there were a significant difference between the two groups(P < 0.01).The blocking rate of serotype 2 AF strain was 51.72% in TSB group and 43.4% in LB group,with significant difference between the two groups(P < 0.05).RA serotype 1SC strain was taken as the test object,and the blocking rates of sterile filtrate samples of chocolate TSP culture suspension made from rabbit,duck and goose blood were75.59%,69.05% and 68.52%,respectively,with significant difference between the former and the latter two(P<0.05).In the FS samples of SC strains recovered from dead ducks,the blocking rate of TSB transmission in the third generation were21.07%,then increased,reaching the peak at 72 hours,but the blocking rates from 24 hours to 96 hours was 32.08%-39.30%,with little change.In the 4th and 5th generation groups,the blocking rates were only 7.97 and 3.81% when the culture time were 12 hours,31.24% and 25.38% when the culture time were 24 hours,and reached the peak value at 96 hours.The results showed that the EPS production of RA in TSB were high,rabbit blood were more beneficial to its production,and its production were affected by ex vivo generation.5.The law of EPSAg production in ducks.14-day-old ducks were artificially infected with RA serotype 1 SC strain,and FS extracted by plasma were detected by AGID for EPSAg.The results showed that all(3/3)of them were positive at 12 hours,and the concentration were 1:2,which rapidly increased to 1:8 after 24 hours and reached 1:16 after 36 hours.The concentration of RA EPS in duck blood were consistent with the dynamic change of blood bacterial load.RA serotype 2 AF strains were negative at 12 hours and 24 hours,the concentration of 36 pih were 1:2,and the concentration of 48 pih increased to 1:8.The dynamic changes of blood bacterial load in this group were consistent with that of RA serotype 1 SC strain,but EPS Ag were negative in the early stage of infection,which showed that there were differences in EPS production between the two strains. |
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