| Tembusu virus(TMUV)infection is a new viral infectious disease in China in2010,which is characterized by a serious decline in the laying rate of laying ducks and neurological symptoms of ducklings.Vaccine immunization is one of the important measures to prevent and control TMUV infection.TMUV attenuated vaccine has become a widely used vaccine in production practice because of its advantages such as less immunization times,rapid immune production and long-lasting immunity.However,some ducks in some duck farms still developed Tembusu virus disease after being vaccinated with TMUV attenuated vaccine.so there are still many doubts about the immune protective effect of TMUV attenuated vaccine in practical use.At present,studies on the immune protective effect of TMUV attenuated vaccine are mainly focused on laying ducks,but there are few reports about the immune protective effect of TMUV attenuated vaccine on ducklings.Therefore,in this study,the genetic variation of the isolated TMUV was analyzed in order to study whether TMUV attenuated vaccine still has the ability of immune protection against epidemic strains.At the same time,the TMUV attenuated vaccine was used to immunize ducklings of different ages through different immune pathways,and whether the immune age and the change of immune pathway could have a positive impact on the immune protective effect of TMUV attenuated vaccine was considered,to provide a theoretical basis for the safe and effective use of attenuated vaccine in the process of prevention and control of Tembusu virus disease.In this study,from 2017 to 2019,suspected TMUV infected ducks were collected from Shandong,Sichuan,Anhui and other regions,and the virus was isolated and identified by cell culture and RT-PCR.A total of 21 strains of TMUVwere obtained.The E genes of the isolates were amplified and sequenced,and their E genes were analyzed by genetic evolution analysis.The results showed that all the21 TMUV strains isolated in this study belonged to Chinese strain ?(TMUV dominant strain),and had close genetic relationship with the previously isolated TMUV located on this branch,with high homology,indicating that there was no obvious variation in the main genes of TMUV,indicating that the TMUV attenuated vaccine still has the ability of immune protection against epidemic strains.1-day-old,3-day-old,5-day-old and 7-day-old Cherry Valley ducklings were immunized with TMUV attenuated vaccine by intramuscular injection and oral administration.The intramuscular injection dose was 0.5mL per animal,and the oral dose was divided into 0.5mL per animal and 0.8mL per animal.The clinical response and survival of ducklings were observed every day after immunization.Cloacal cotton swabs were collected at 1 d,3 d,5 d,7 d,9 d,11 d,13 d and 15 d after immunization,and fluorescent quantitative RT-PCR was used to detect the detoxification of each group.At the same time,blood samples were collected after one week of immunization,and then blood samples were collected every 3 d and indirect ELISA detection was used to detect the changes of antibody levels in serum.The results showed that there was no obvious abnormal reaction after oral and intramuscular injection of TMUV attenuated vaccine in ducklings of different immune days.at 5 days old,ducklings immunized with TMUV attenuated vaccine by oral or intramuscular injection could quickly produce high level of antibody.5-day-old Cherry Valley ducklings were divided into immune infection group,non-immune infection group and control group.When ducklings were raised to 15 days old,the ducklings were inoculated with virus solution(DTMUV-SDLC)at the dose of 1.0mL per animal,and the control group was injected with the same dose of aseptic PBS.The clinical response and survival of experimental ducks were observed every day after infection.At 7 d,14 d,21 d and 28 d after infection,3birds in each group were randomly selected and killed,and the pathological changes were observed and compared.At the same time,blood samples were collected at 3 d,6 d,9 d,12 d,15 d,18 d,21 d and 24 d after infection,and the changes of serumantibody levels were detected by indirect ELISA detection.The results showed that compared with the ducklings in the non-immune infection group,the ducklings in the immune infection group did not show any clinical symptoms,no death,only slight congestion in capillaries in the brain,dark red in the spleen,and no pathological changes in other organs;in addition,after 5-day-old ducklings were immunized with TMUV attenuated vaccine by oral and intramuscular injection,they had 100% protection against the attack of virulent DTMUV-SDLC,and achieved a good immune protective effect. |
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