Investigation Of Avian Tembusu Virus In Jiangsu、Shandong And Sichuan Province And Development Of A New Elisa By Using Sheep Anti-Goose IGG

In order to know the TMUV infection in avian, samples collected from ducks, chickens, geese were investigated of TMUV antibody and virus strains were isolated. From May.2012to Jan.2013,221duck serum (yolk) collected from Jiangsu and Sichuan province had been detected by using indirect ELISA. Results showed that the seropositive rate of Jiangsu province is78.5%(142/181)and Sichuan province is0(0/40). The seropositive rate of Shaoxing laying duck is the highest with90.9%(100/110); the corresponding part of Maple breed duck is65.6%(40/61) and Cherry valley breed duck is20%(2/10).The antibody level of duck groups in Nanjing were surveillanted. Result showed that the level of antibody is low in May and January of next year and is high in September.During Oct2012and Jan2013,84chicken serum (york) and10suspicious TMUV infected broilers’s tissue samples were collected from Jiangsu province. Results showed that the seropositive rate of chicken is57.1%(49/84) among which Liyang is84.6%(11/13), Nanjing is57.7%(15/26) and Danyang is51.1%(23/45). The seropositive rate of egg chicken is the higher one with66.7%(26/39) broilers is51.1%(23/45). Ten tissue samples are all negative for TMUV shown by nested RT-PCR so the TMUV pathogen positive rate is0(0/10).From Jun.2012to Mar.2013,317goose serum and43suspicious TMUV infected geese tissue samples collected from Jiangsu, Shandong and Sichuan provinces were detected by indirect ELISA using anti-duck and nested RT-PCR respectively. Results showed the seropositive rate of Jiangsu is55.8(144/258), Shandong is23.1%(9/39) and Sichuan is0(0/20). The seropositive rate of meat goose is75.5%(102/135), breeding goose is34.1%(42/123) and overfeeding goose is23.1%(9/39). Two tissue samples were detected positive for TMUV among all the43samples so the TMUV pathogen positive rate is4.65%(2/43). Part of the sequences of NS5gene of two isolated strains were analyzed. Results showed both of the TMUV strains shared a high identity of more than99%with other recently isolated TMUV.Purified recombinant E protein as coating antigenf the TMUV strains shared a high identity of more than99%with other recently isolated, then geese serums were added, following adding sheep anti-goose IgG, finally after adding HRP marked anti-sheep color was developed. What’s more, ELISA reaction conditions had been optimized. Results showed the best antigen coating concentration is4μg/mL; best antibody dilution is1:28; optimal antigen coating condition is incubated overnight of4℃; the best sheep anti-goose incubation time is2h and concentration is1:400; the best HRP anti-sheep incubation time is1h and concentration is1:8000; cut-off value is associated with the X+3SD. Sensitivity and reproducibility were all tested to be fair. This new ELISA test was applied on detecting80goose samples from Shandong and Jiangsu province. Results showed TMUV seropositive rate in both provinces is65%(52/80).The seropositive rate of the same80samples detected by using sheep anti-duck IgG turned out to be52.5%(42/80). The coincidence seropositive rate of these two ELISA results is80.8%(42/52) and coincidence seronegative rate is73.7%(28/38).