The Establishment Of RT-PCR Method For The Rapid Diagnosis Of IBD And Isolation And Identification Of VvIBDV In Anhui

Infectious bursal disease (IBD) caused by infectious bursal disease virus is anacute, highly infectious, lymphocidal disease of young chickens.The disease has beenrecognized in virtually all major poultry producing areas worldwide and is consideredeconomically significant because of its ability to induce a profoundimmunosuppression in chicken with increased susceptibility to other infectious agentsand diminished vaccine response. With the advent of vvIBDV and vIBDV, IBD ismore prevalent than before in China. In order to develop an available method todiagnose IBD, the primers lied in the overlapping area of VP5 and VP2 will bedesigned and a reverse transcription polymerase chain reaction (RT-PCR) which is arapid, specific and sensitive method for diagnosing IBD will be developed.Unfortunately, IBD breaks out more frequently than before, on occasion, it isfulminant and mortality is very high, but the research on it is not relatively enough inAnhui province. In order to quest for its causation, the three IBDV strains will beisolated from bursal tissure of infected chickens and pathogenicity and the VP2hypervariable region sequences will be researched. Firstly, according to the sequences of published strains of 3 pathotypes ofInfectious bursal disease virus, the primers lied in the overlapping area of VP5 andVP2 were designed and a reverse transcription polymerase chain reaction (RT-PCR)was developed. The assay of all 4 referential strains which including pathotypescIBDV, vvIBDV, and vIBDV can amplify the products of 222bp, but not theNewcastle disease virus, Infectious bronchitis disease virus, Marek's disease virus andColibacillosis, which can result in other chicken diseases. The sensitive experimentindicated that B87 5fg cDNA that was diluted to as low as 1/100 was still detected bythe RT-PCR. Simultaneously, 25 samples, which were collected from Anhui,Shangdong and Sichuan were detected by using developed RT-PCR and AGP. Allsamples were positively detected by the RT-PCR, while 23 samples were positivelydetected by the AGP. Virul isolation had been done for the 2 negative samples (SZ1and FY1)by using CAM and CEF and inoculating chickens. SZ1 led to the typicalpathological changes of embryos, chickens and CEF cells for IBDV, and detectedallantoic fluid by the RT-PCR, the result was positive.However, FY1 didn't produceany pathological change of them, and detected allantoic fluid by the RT-PCR, theresult was negative.Secondly, by using embryo inoculation test, and electron microscopic observation,we got 3 pure IBDVs from 3 samples, which were collected from CH, JG and QJ. Andthey were designated as CH03 strain, JG04 strain and QJ04 strain respectively. Inorder to study their pathogenicity, four-week-old chickens inoculated with 3 strainsdeveloped severe clinical disease at morbidity of 100% and mortality of 92%, 83%and 67%. And the most characteristic primary gross lesions were severe bursalhaemorrhage and thymic atrophy, as well as the bone marrow with fatty change.ELD50 of virus were 10- / 0.2ml﹑10- 6.8 5.4 /0.2ml﹑10-4.6/ 0.2ml. All of those showedthat 3 isolates had been very virulent IBDV strains. Thirdly, the VP2 hypervariable region(AccI-SpeI)of 3 isolates(CH03 strain,JG04 strain and QJ04 strain) from Anhui province was amplified byRT-PCR/Nested-PCR. Three segments were cloned into pMD18T vector, sequencedand analyzed. By using the software of Dnastar and referencing to others IBDVstrains' published sequences in Genebank, we analyzed molecular characteristics oftheir VP2 hypervariable region and constructed their phylogenic trees. The resultswere as follows: Amino acid position 249(K) and 254(S), which turned out variantIBDVs, and our 3 isolates existed 249(Q) and 254(G) at positions, which proved thatantigenicity was invariant.The serine-rich heptapeptide sequence"SWSASGS'' wasconserved among our strains. 3 IBDV isolates exist 279(N-D) and 284(T-A), whichare necessary to their virulence. Four amino acid conserved in very virulent isolatesreported earlier at 222 P-A, 256V-I, 294L-I and 299N-S were conserve...