Dynamic Analysis Of Duck Enteritis Virus Virulent Strain And Its Attenuated Strain In Experimentally Infected Ducklings

Duck Viral Enteritis(DVE), also known as Duck Plague(DP), common in ducks, geese, andother en echelon poultry, caused by duck enteritis virus (DEV), is an acute, septic and highly contactinginfectious disease. The main clinical symptoms of DVE are vascular injury, digestive tract mucosahemorrhage necrosis, lymphoid organs and substantive organs in degenerative lesion. As its highmorbidity and mortality rates, DVE has brought great damage to duck industry. At present, the mainmeasure of prevention and control is vaccine immunity. As the study about distribution of virus inanimal body and proliferation rule is an important basis to clarify the virus pathogenic mechanism andthe immune mechanism, so it is very necessary to build a molecular biological method to study thedynamic distribution of virulent and attenuated strain in infected duck body, as to elucidate thepathogenesis of DEV virulent strain and provides the basis for the reasonable and effective preventionof DVE.According to the sequence difference between DEV CSC virulent strain and its attenuatedstrain DEV p80, in this study,two kinds of SYBR Green Ⅰreal-time fluorescent quantitative PCR methods were developed,one for detection DVE virulent and attenuated strain,the other for distinguishing the virulent strain from its attenuated virus.The results showed that both the methods had good linear relationship betwwen initial templates and Ct values,and the two coefficientcorrelation of the standard curve were1.0.The solubility curve showed the two methods werespecific,and the TM value respectively were80.5℃,81.5℃.The specificity of two assays were proven based on no amplification by detecting DNA from normal duck tissues,duck viral hepatitis virus,newcastle disease virus,Pasteurella, Riemerella anatipestifer et al. The two assays had coefficient of variations less than2.5％for both intra-and inter-assay.The sensitivity of SYBR Green Ⅰreal-time fluorescent quantitative PCR method for detection DVE virulent and attenuated strain had a detection limit of9.072copies of pMD18T-gD,7.42copies of DEV CSC DNA(1MLD),40.5copies of DEV P80DNA(100.5TCID50),while the other one had a detection limit of4.77copies of pMD18T-gI,4.23copies of DEV CSC DNA(1MLD). According to the results, both the two methods had good specificity, sensitivity and repeatability.The kinetics of virus loadwas examined in ducklings infected with DVE CSC virulent strain and DEV attenuated p80strain.The samples were collected from thymus, spleen, bursa of Fabricius, heart, liver, lung, kidney,pancrease, duodenum, ileum, caecum, colon, rectum, brain, trigeminus, serum, cloacal swab.DEV DNA copies in samples were detected by the general type of SYBR Green I real-time fluorescent quantitative PCR method for DEV virulent and attenuated strain. The results showed that DEV CSC DNA were firstly detected in all the samples at six hours after inoculation andthe DNA copies subsequently continued to rise till the death of all the ducklings infected withDEV CSC virulent strain five days after inoculation. The DNAcopies ofDEV CSC strain in colon(1013.26)of dead ducklings was highest,and then came to bursa of Fabricius(1012.12), caecum(1011.92)andliver (1011.82). The results above showedthat the virulent strain CSC had broad tissue tropism on immune organs, nerve tissue and digestive system of infected ducks. The DNA copiesof DEV p80strain in tested samples after inoculation increased firstly and then decreased on the whole, and the ceiling copy numbers of various organs were different, maximum in thymus (108.82) and then came brain (108.14)and liver(107.97). Compared with CSC strain, the distributionof DEV p80strain in diverse tissues and organs of immune ducks was quite similar, but DEV p80strain DNA copy numbers in tissueswere significantly lower than CSC strain,The DNAcopies of DEV p80strain in spleen,pancrease，digestive tract and cloacal swab fell by approximately106times than maximum copies of DEV CSC strain，which made the most obvious difference，while bursa of Fabricius，liver respectively fell by105，104times，which took the second，there was no significant difference between copies of DEV CSC strain and copies of DEVp80strain in thymus，brain，trigeminus，The DNA copies of DEV p80strain in them only fellby101~102times than DEV CSC strain.Finally, cloacal swabs of ducklings immunized with DEV p80strain were assayed through thedifferential type SYBR Green I real-time fluorescent quantitative PCR methodafter challenge with DEVCSC. The result showed that the dynamic proliferation regularity has significant difference. Copynumbers of DEV CSC virulent strain continue to rise in the control ducks, while copy numbersin theducks vaccinated withDEV p80strain stayed low, far lower than controls. We could conclude thatducks vaccinated with DEV p80couldprevent from a virulent strain, and the copies in cloacal swab ofthe vaccinated ducklings fell by105times than copies of the controls. DEV p80attenuated strain ispotential to develop into a efficient vaccine, for the prevention and control of the duck plague.