Establishment Of Immunoperoxidase Monolayer Assay For Detecting The Serum Antibody Of Peste Des Petits Ruminants

Peste des petits ruminants(PPR) is caused by a Morbillivirus that belongs to the family Paramyxoviridae. PPR is an acute, highly contagious and fatal disease primarily affecting goats and sheep. In1942, PPR occurred in Western Africa and then outbreak continutes to spread in other countries. In 2007, China’s first outbreak of the PPR epidemic wasin Tibet, and from 2013 to 2014, the PPR outbroke in 19 provinces of China, which caused huge losses for the livestock breeding in our country. In April 2015, the animal health expert meeting held in Abidjan, the capital of Ivory Coast, agreedto try to eliminate the PPRV in the world. Therefore, a comprehensive study about etiology, epidemiology, diagnosis and prevention and control measures of PPR should be carried out as soon as possiable.Given this, a rapid sensitive diagnostic technique method will produce far-reaching social and economic implications for the prevention and control of PPR.Currently, there are many diagnostic methods for PPRV. VNT(virus neutralization test) and c-ELISA are main serological detection methods. However, VNT needs long time and requires higher test conditions; c-ELISA has the disadvantages of high price and complex preparation. Immunoperoxidase monolayer test assy(IPMA) is a rapid, sensitive, specific and simplediagnostic method, has been used to detect multiple pathogens in clinical samples. But the IPMA detection methods for PPRV have not been established both at home and abroad, therefore,the establishment of IPMA has some significance for the monitoring and prevention of PPR.In order to establish the IPMA for PPR, this study firstly established theBHK-21 cell line which stably expressinggoat SLAM(BHK-gSLAM). In comparison with Vero cells, rPPRV/GFP infection experiments showed that PPRV could replicate well in this cell line and distinctive syncytium was produced. Western blotting showed thatBHK-gSLAM could stably express SLAMprotein at least 20 generations. On this basis, the IPMA for detecting the serum antibody of PPR was established. During the IPMA plates preparation, when the BHK-gSLAM cells and Vero cells were infected with the same dose of virus, CPE formation time inBHK-gSLAM cell is significantly earlier than that in Vero cells. Furturemore, the AEC staining results wereeasier to be observeddue to the large syncytia formation inBHK-gSLAM.Finally, the BHK-gSLAM was used for the IPMA plates preparation. In this study, IPMAconditions was optimized. The appropriate virus dose was 104 TCID50/mL and 72 hlater the IPMA reaction plateswere fixed. The optimal dilution of serum and secondary antibody working concentration are 1:10 and 1:5000, respectively. Finally, 198 goat serum(including clinical samples and samples after vaccind with PPR attenuated vaccine strain) were detected with IPMA, and VNT as a control method. The sensitivity and specificityof IPMA as compared with VNT was 91% and100%, respectively. The overall agreement quotient between results of the two tests was 95.5%.These results fully approved that IPMA method established in this study was a reliable method with good specificity and sensitivity. The detection time was only three hours, it would be an adequate substitute for the VNT for assessing herd immune status and for epidemiologic surveillance.