Isolation,Identification And Pathogenicity Of A New Type Duck Reovirus

In recent years,a disease characterized by spleen necrosis has emerged in China's meat duck breeding areas.The disease mainly causes a mental state of the ducks,the drinking and feeding intakes are declined and accompanied by a certain mortality.After spleen necrosis,the duck's immunity is declined,and it is easy to cause higher mortality due to secondary infection.Resistant to duck development hindered,slow growth,become a stiff duck.At present,most researchers believe that the new type duck reovirus(NDRV)is the main pathogen causing this disease.For the characteristics of the disease are at onset small age,developing rapidly,and it is easy to spread quickly.It is urgent to establish a rapid,accurated and reliabled method for early stage of the disease's qualitative and quantitative diagnosis.In this study,a duck new reovirus(NDRV)was isolated from the necrotic spleen of ducks,and the isolated strains were sequenced and study its pathogenic.A TaqMan quantitative RT-PCR method was established for this disease,and monitored the distribution in different tissues and organs of infected ducks about NDRV,the detoxification and the viremia of the virus.It provide a theoretical basis for NDRV prevention and control.A pair of primers and a specific probe were designed based on the sequence of NDRV S2 gene,and the amplification length was 85 bp.The amplified fragment was ligated into pMD18-T vector to construct a recombinant plasmid.After screening and sequencing,the dilution was used as a standard.After optimizing the reaction conditions,a standard curve was constructed to establish a TaqMan method for quantitative detection of NDRV.The standard curve of this method has a good linear relationship.The linear regression equation is y=-3.3468x+41.681,and the correlation coefficient R~2 is 0.9988.The method had high sensitivity.The minimum detection template concentration is 10 copies/?L,which is 1000times that of ordinary PCR.Only NDRV can be detected,and there is no cross-reaction with other viruses,it is showed that this method's specificity was high.The inter-assay and intra-assay coefficient of variation is below 3%,so the repeatability is good.The above experiments show that the TaqMan quantitative RT-PCR method method established in this study has high sensitivity,specificity and reproducibility,and can be used for clinical detection of NDRV.One day old healthy cherry valley ducklings(180)were randomly divided into 3groups(the muscle group,the oral group and the control group),60 per group.The oral group and the muscle group were inoculated with 0.2 mL of virus solution at 1 day,and the control group was inoculated with 0.2 mL sterile saline,respective.The clinical symptoms and necropsy changes of the ducklings were observed and recorded.The weight was measured every 2 days to monitor the changes of body weight.The cloaca cotton swabs and anticoagulation were collected to detect the detoxification and viremia of the ducks.At the 12h,1 d,2 d,4 d,6 d,8 d,10 d,12 d,14 d,16 d,18 d,21 d,24 d,27 d,30 d after the attack,3randomly selected groups were used to prepare paraffin sections for the diseased tissues and organs.The histopathological changes were observed and the tissue was measured for viral load.The results showed that the spleen enlargement,hemorrhage and necrosis were observed in the ducklings of the experimental group.The weight gain of the ducklings in the experimental group was slow,and the oral group was the most significant.The results of the cotton swab detoxification showed that both groups had detoxification in the early stage of infection.The results of virus content changes in blood showed that the virus was detected in both groups at the early stage of infection,and the content change was basically consistent with the cotton swab detoxification rule.The virus tissue load results showed that after artificial infection of SDYC strain,the virus can be detected in the heart,liver,spleen,lungs,kidneys,pancreas,duodenum,stomach,bursa,thymus,and brain.The organs with higher virus content in the two groups are the spleen,thymus,and bursa,which can be used as the preferred organ for the disease's detected.One day old healthy Rose 308 broiler chicken and SPF chicken were randomly divided into muscle group,oral group and control group.Oral group and muscle group were inoculated with 0.2 mL of virus solution at 1 day,and control group was inoculated with 0.2mL of sterile physiological saline.Observe the clinical symptoms and necropsy changes after chick infection.The results showed that SPF chicks and broilers showed mental depression and death in the muscle group.The mortality of the muscle group of SPF chicks was 100%,and that of broiler muscle group was 71.4%.There was no significant change in the oral group and the control group.The necropsy lesions of the dead chicks were generally consistent,including a large amount of ascites in the abdominal cavity,swelling of the liver and spleen,hemorrhage,and large area necrosis.It is showed that NDRV has the possibility of infecting flocks in production,and it is necessary to strengthen biosafety prevention.