Establishment Of Fluorescence Quantitative RT-PCR For Detection Of New Duck Reovirus And Evaluation Of The Immunity Efficacy Of Inactivated Vaccine

Since 2017,the incidence of Novel duck reovirus(NDRV)has increased rapidly in my country,and the incidence area has also been expanding.The age of onset is mainly in the range of 4d-26d,and the incidence rate is 5%-80%.Among them,the mortality rate can reach 20%.Generally,the younger the age,the higher the morbidity and mortality.In this study,primers were designed based on the conservative sequence of the unique non-structural protein P18 gene of the new duck reovirus,and the NDRV SYBR Green I fluorescent quantitative RT-PCR detection method was successfully established.The inactivated vaccine was prepared with the NDRV-SY strain and the vaccine immunization was carried out.Effectiveness evaluation research has laid an important foundation for the prevention and control of the disease.This research includes the following two parts:1.Establishment and application of novel duck reovirus P18 protein gene fluorescence quantitative RT-PCR detection methodIn this study,a quantitative RT-PCR method was established with one pairs of specific primers designed based on the conservative sequence of the unique non-structural protein P18 gene of novel duck reovirus(NDRV).239 clinical samples were detected by this method and ordinary RT-PCR methods,and the positive detection rates of the samples were 23.85%(57/239)and 17.57%(42/239),respectively.The coincidence rate of the two methods was 93.7%.The detection rate of fluorescent quantitative PCR method is significantly higher than that of ordinary RT-PCR method.The 14-day-old ducklings were artificially infected with the NDRV-SY strain with a dose of 103ELD50,collected Cloaca anal swab samples at different times,and the detoxification status was monitored by fluorescence quantitative RTPCR.The results showed that the artificially infected ducklings with NDRV can detect the virus excretion from the cloaca for 48 hours,and the duration is up to 20 days,and it was found that there were 2 peaks of excretion on the 5th and 11th day after the virus infection.The test results showed that the established NDRV fluorescent quantitative RT-PCR detection method has good specificity and high sensitivity,and can be used for detox monitoring of NDRV infection,as well as early diagnosis of clinical infection and epidemiological investigation.2.Preparation of novel duck reovirus inactivated vaccine and evaluate its immune efficacy(1)Preparation of NDRV inactivated vaccine.The BHK cells of the NDRV-SY strain were used to adapt to the virus to expand the culture,and then inactivated by formaldehyde.Use white oil and Spencer-80 as the oil phase,mix the inactivated NDRV-SY strain virus liquid and sterile Tween-80 as the water phase,and mix and emulsify according to the 3:1 ratio of the oil phase to the water phase.Inactivated vaccine into oil adjuvant.(2)The NDRV duckling challenge model was established.With different doses of NDRV-SY strain duck embryo tissue poison,the 17-day-old and 24-day-old NDRV antibody-negative ducklings were challenged by subcutaneous injection,and 8 test ducks were selected 5 days and 7 days after the challenge,autopsy to observe the liver and spleen tissue lesions,and select the obvious lesions for histopathological examination.The results showed that for 17-day-old ducklings,the lesion rate of liver and spleen tissue was 100%(8/8),at 5 days after challenge with 104ELD50,and the rate at 7 days after challenge with 103ELD50 was 87.5%(7/8);24-day-old ducklings,the rates were 75%(6/8)and 87.5%(7/8)at 5 days and 7 days after challenge with 104ELD50 dose.Therefore,a preliminary challenge model of NDRV-SY strain was established:17-day-old NDRV antibody-negative ducklings were challenged with duck embryo tissue poison of NDRV-SY strain at a dose of 103ELD50,and liver of ducklings could be observed at necropsy 7 days after challenge,the spleen has obvious tissue disease.(3)Evaluation of immune efficacy of NDRV inactivated vaccine.The NDRV inactivated vaccine was immunized with 3-day-old NDRV antibody-negative ducklings and the immune efficacy of the vaccine was evaluated through three aspects:antibody detection,post-immune challenge lesion inspection,and detox monitoring.ELISA antibody detection:3-day-old NDRV antibody-negative ducklings were divided into 4 groups.Three groups were immunized with 0.25 mL,0.5 mL,and 1.0 mL doses of vaccine respectively,and the other group was injected with 1.0 mL of normal saline as a control.Blood was collected 7 days,14 days,21 days,28 days,and 35 days after immunization to separate the serum,and the serum antibody level of the test ducks was detected by indirect ELISA method.The results showed that the positive rate of ELISA antibody for ducklings in the 0.25mL group was 18.75%14 days after vaccination,and 43.75%and 50%in the 0.5mL/1.0mL group;the ELISA antibody level increased significantly on the 21st day after immunization,and the ELISA antibody positive rate in the 0.25mL group was 56.25%,and the 0.5mL/1.0mL group reached more than 80%.From 28 days to 35 days after immunization,the 0.5mL/1.0mL group ELISA antibody positive rate was 100%.Immune protection rate and detoxification test:14 days after immunization,16 ducklings in each group were randomly selected for challenge with the NDRV-SY strain,the challenge dose was 103ELD50/bird,and 8 ducks from each group were randomly selected 7 days after the challenge.The ducklings were examined by necropsy to observe the pathological changes of liver and spleen tissue.The results showed that when the immunization dose of the NDRV-SY strain inactivated vaccine was 0.5 mL,the immune protection rate could reach more than 80%.Cloacal swab samples of ducklings were collected at different time points after the immune challenge,and the detoxification status was monitored by the established fluorescent quantitative RT-PCR method.The results showed that the amount and duration of detoxification in the 0.5mL/1.0mL immunized group were significantly lower than that of the 0.25mL immunized group and the non-immunized control group.According to this,the NDRV-SY strain oil emulsion inactivated vaccine(107.83TCID50/mL)prepared in this research has a good immune protection effect when the immunization dose is 0.5mL,which lays the foundation for the subsequent NDRV vaccine research and development.