Effect of E.wenyoni on Blood Biochemical Parameters of Cows and Establishment of Sandwich ELISA、PCR for Detecting E.wenyoni
|School||Agricultural University of Hebei|
|Course||Preventive Veterinary Medicine|
|Keywords||Cow E.wenyoni Biochemical parameters Sandwich enzyme-linked immunosorbent assay PCR|
Cow with natrual infection of E.wenyoni were used as experimental animals.Blood sample were collected asepsisly with the nature,and were observed through direct microscopy and Giemsa stained blood smears. It was found that E.wenyoni were single or aggregate that existed in blood plasma or attached to erythrocyte which took on deformitie. E.wenyoni that attached to eryhrocyte with Giemsa staining.was purple red.The content of NO and MDA,the activity of SOD in the blood were determined. The results were as follows:with the enhancing of the infection,the content of NO and MDA increased,and the activity of SOD decreased.These findings indicated that the changes of the three kinds of biochemical parameters was relevant to infection of E.wenyoni.E.wenyoni antigen was collected from naturally infected blood through washing by PBS-T solution,centrifugation at 4°C and ultrasonic method.The hyperimmune serum of rabbit anti-E.wenyoni was prepared by immunization in rabbit with purified E.wenyoni.Mainly immunoglobulinG(IgG) of rabbit anti-E.wenyoni was purificated by caprylic acid-ammonium sulphate precipitation,the result of PAGE suggested that it is one simple,applied and efficient method for purification of IgG from serum.The sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for detecting E.wenyoni antigen was established.The results showed that the most suitable concentration of coating antibody was 156 μg/mL,the most suitable dilution of enzyme labeled rabbit-anti-bovine antibody was 1:400 and the minimum detection of the content of antigen reached 6.64 μg/mL.The pure antibody of rabbit anti-E.wenyoni could react on the whole blood and erythrocyte with E.wenyoni,the most suitable dilution was 1:160 and 1:40 which could be applied to detect E.wenyoni antigen in the blood.Through the blocking test,cross test and duplication test we can see clearly that the method of sandwich ELISA is very specific,sensitive, efficient and is suitable for group detection.The assay was applied to detecting E.wenyoni in 222 blood samples fromHebei.,E.H wenyoni-carrier rates of cow was 91.0%.It proves that E.wenyoni extensivelyexists in our province .The investigation shows the epidemiology of E.wenyoni in Hebeiand provides the stable basis of prevention and control of its spreading.A pair of primers was designed and synthesized according to the conservative sequence ofE.wenyoni 16 S rRNA.A 985bp fragment was amplified by using polymerase chainreaction. The amplified fragments with the expected size were identified by EcoR I restriction digestion.The crossing-reaction,specific-reaction and duplicate-reaction indicated that the method of PCR is specific,sensitive and quick.It is a effective way to be used to diagnose E.wenyoni from the whole level.