| Riemerella anatipestifer(RA)disease,also known as duck infectious serositis.The disease was a common acute septic epidemic disease in ducklings,young turkeys and other poultry.Its main manifestations were cellulosic pericarditis,perihepatitis,air sacs,salpingitis and meningitis.The disease had a high mortality rate and a high elimination rate,which is extremely harmful to ducks.Recently,the problem of drug resistance had became increasingly serious.Monoclonal antibodies were highly specific,had no drug resistance problems of drug resistance and residues.The experiment prepared a monoclonal antibodies by whole-bacterial immunoassay.In order to evaluate the effect,the animal test was completed.That provides technical support for the prevention and treatment of RA.The content of the test was carried out from the following aspects.1 The isolation and identification of bacterialIn this study,bacteria were isolated from diseased materials for differential culture.Staining microscopy was completed.The bacterial gene was amplified by PCR with universal primers,and the amplified products were sequenced and aligned with BLAST to construct a phylogenetic tree.Drug susceptibility test and biochemical characterization were completed.Bacterial serotypes were identified by slide agglutination test.Infecting ducklings with pure bacterial culture to test regression pathogenicity.The results show that the isolation was RA.glucose,lactose,maltose,fructose,mannitol can not be fermented,hydrogen sulfide and gelatin puncture results were negative,MR and VP separation experiments were negative,hydrogen peroxide can be decomposed.Bacteria was highly sensitive to streptomycin,moderately sensitive to amikacin,and low sensitive to amoxicillin,ampicillin,kanamycin,cotrimoxazole,ciprofloxacin,gentamicin,polymyxin,and tetracycline.Bacteria were RAⅡ type.The bacteria can replicate cases of typical serositis.2 Preparation of m Abs against RARA and Freund’s adjuvant were mixed and emulsified as a vaccine.The SPF-level BALB/c mice were immunized four times with the vaccine and immunized every two weeks.The last immunization was intraperitoneally injected with the same dose of antigen without adjuvant.Three days later,if the serum titer of the mice was higher than 1:6400,the spleen cells were fused with the viable myeloma cells for cell fusion,cloning,and subcloning.The obtained positive cells were subjected to cryopreservation stability test,secretion stability test,karyotype analysis,specific identification by E.coli and Salmonella as reference strains,identification of monoclonal antibody subclasses by kit,and Western blotting.The results showed that three positive hybridoma cell lines were obtained,which were named as 3A8,3D11,and 4D1.These cells grew well before and after cryopreservation,and the OD value of the cell supernatant was stable.The indirect ELISA results showed that 3D11 were Ig G3 isotype,and 3A8 and 4D1 were Ig M isotypes.With good specificity,these done not cross-react with E.coli and Salmonella.Western-blot reslults showed that m Abs 3A8 and 4D1 could bind to protein bands of approximate 55 KD,while 3D11 could not.3 Protection test of m Abs against RAEighteen 10-day-old ducklings were divided into three groups according to each group of six.One group was infected with artificial infection by 2×109CFU/m L RA0.1m L and normal saline 0.1m L,as the infected group.One group was infected with artificial infection by 2×109CFU/m L RA 0.1m L and antibody 0.1 m L,as a protection group.The last group was injected with 0.2 m L physiological saline,as a blank control group.Observed for 7 days and dissected all ducks after 7 days.The results showed that the infection group had more deaths and faster death rates than the protection group.There was no significant change in the blank group.The necropsy changes showed that the tissues and organs of the protection group were slightly damaged,and the injury of the infection group was obvious.The pathological changes of the infected group and the protective group were similar.Both showed tissue edema,hyperemia,myocardial cell waxy necrosis,hepatocyte cell structure disorder,vacuolization,and necrosis of brain cells.The results indicated that monoclonal antibodies had the effect of delaying onset,alleviating symptoms and reducing mortality.The experiment isolated RAⅡ type,which belonged to the main serotype of China.The bacteria were highly sensitive to streptomycin and moderately sensitive to amikacin,to amoxicillin,ampicillin,kanamycin,cotrimoxazole,ciprofloxacin,gentamicin,polymyxin,Tetracyclines were all low-sensitivity and realistically consistent.The test results proved that the monoclonal antibodies had certain protective effects and practical value.The using of mabs to control RA,the test put forward a theoretical basis for this. |
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