Serological Investigation Of Infectious Bovine Rhinotracheitis In Beijing Regions And Development Of An Indirect ELISA For Detecting Antibodies Against IBRV

To understand the epidemic situation of infectious bovine rhinotracheitis(IBR) in Beijing cow herds.2582 serum samples collected from cows in various districts of Beijing dairy were detected using commercial IBR gE antibody test kit.The surveillance results showed that seropositive ratios for antibody against IBR virus were varied from regions.The rates of positive cows were distributed individually in Changping 23.6%(245/1037),Fangshan 100%(50/50),Yanqin 50.63%(202/399),Shunyi 60%(9/15),Fengtai 30.03%(164/546) and in Miyun 43.46%(232/535).Totally,the average rate of IBRV seropositivity in Beijing was 34.93%(902/2582).It suggested that IBR prevalence were serious in current cow herds of Beijing and the prophylaxis measure should be taken as soon as possible.To develop a diagnosis method for IBRV infection,IBRV was propagated on Madin-Darby bovine kidney(MDBK) cell cultures, and purified IBRV was gradient centrifugation.An indirect enzyme linked immunosorbent assay(ELISA) was successfully established using above purified IBRV as coating antigen.The optimal concentration of coating antigen and dilution of test sera were determined by checkerboard titration on a 96 well ELISA plate. Purified IBRV was diluted to 20μg/ml when coating immunomicrotiter plates and standing at 4°C overnight.The plate was washed with a standard washing buffer PBST and blocked with 3% bovine serum albumin(BSA) in washing buffer for 2h at37°C.100μl of serum with dilution of 1:80 in blocking buffer was added in coated well for1 h at 37°C.Equal amount of 1:20000 dilution of anti-bovine Ig G peroxidase conjugate was incubated for 1h at 37°C.3,3,5,5-tetramethyl benzidine(TMB) was added as substrate and reacted for 10 minutes at room temperate in dark box.The cut off OD value between positive and negetive was 0.240.Specificity test of this ELISA showed only sera from cows infected with IBRV could be positive, rather than from cows infected with BVDV,Bovine leukemiavirus and Bovine paratuberculosis bacteria etc.Sensitivity of the ELISA was determined to be 1:640 dilution of reference positive sera from IDEXX kit.When compared with the virus neutralization test,the specificity of the indirect ELISA was 100%(23/23),the sensitivity was 82.76%(24/29).Good reproducibility of our IBR ELISA was verified by coefficient of was less than 10% either in same plate or different,the concordance between this indirect ELISA and IDEXX kit showed positive coincidence rate was 87.7%(228/260),and negative was 98%(196/200).Taken together,this indirect ELISA is valuable to be a tool for surveillance of infection of IBRV,in place of currently exotic IBR commercial kits with expensive costs.