The Establishment Of Indirect Elisa Method For The Diagnosis Of Cerebral Coenurosis With TmAdh3 Recombinant Antigen

Cerebral coenurosis is a serious zoonotic parasitic disease in sheep,gosts,yaks and other ruminants caused by the parasitism of Taenia multiceps metacestode Coenurus cerebralis in the brain and spinal cord,which lead to encephalitis and neurological symptoms in infected hosts,or even death in sometimes.It is difficult to diagnose in early infection stage,but it is hard to treat and cause animal death when it is diagnosed because of irreversible injure for nervous system,which cause serious economic loss to animal husbandry.So,it is necessary to establish early diagnosis method for cerebral coenurosis.In the present study,rTmAdh3 was used as the diagnostic antigen.rTmPKA-C1,rTm7,and rTmGST were used as the reference antigen for indirect ELISA development.Firstly,primers were designed according to the Tm7 and TmGST genes and the recombinant expressional vectors pGEX4T-Tm7 and pET30a-TmGST were constructed,transformed into host bacteria to express recombinant protein.Meanwhile,the recombinant bacteria of pEX30a-TmPKA-C1 and pGEX4T-TmAdh3 which were constructed in the laboratory were induced.The recombinant proteins TmAdh3(39.0 ku),TmPKA-C1(42.0 ku),TmGST(30.0 ku)and Tm7(33.0 ku)were successfully expressed.The western-blot results showed that rTmAdh3 could distinguish the serum of naturally infected sheep from that of healthy sheep more obviously than rTmPKA-C1,rTmGST and rTm7.rTmAdh3,rTmPKA-C1,rTm7,and rTmGST were used as diagnosis antigens to establish ELISA.After comparison analysis the results showed that the rTmAdh3 is more distinct than other proteins in serum detection.The sensitivity is 83.3%(15/18)and specificity is 94.4%(17/18)in indirect ELISA established by recombinant TmAdh3.As a diagnostic antigen,the recombinant protein had some reactivity on the serum of sheep after 14 days infected with C.cerebralis(1/9),and a significant reactivity on the serum of sheep after 21 days infected with C.cerebralis(6/9).All the experimental infected sheep after 28 days infected were tested positivity(9/9).In conclusion,the indirect ELISA method for diagnosis of cerebral coenurosis in sheep using TmAdh3 recombinant protein in this study has high specificity and sensitivity.This ELISA has the value of early diagnosis of cerebral coenurosis,which can be applied to epidemiological investigation and animal quarantine,and lays a foundation for prevention and control of cerebral coenurosis in sheep.