Studies On The Method And Its Application Of The Specific PCR And Indirect ELISA Detecting The Goose Parvovirus And Its Antibody

Goose plague (GP) was discovered first in China by Fang Ding-Yi at Yang Zhou in 1956. The virus was isolated from the gosling using the embryonated goose eggs in 1961, and named Goose plague virus. Later, it was nominated Goose parvovirus (GPV). Four to eight weeks old gosling and Muscovy duckling were infected GPV, and the transmission rapidly with more than 90% mortality, which was became most important disease to waterfowl. GP were reported in many countries including Soviet Union, Germany, Hungary, Netherlands, France, Britain, Italy, Israel, Yugoslavia, Vietnam and Japan. GPV was reclassified in Parvovirinae family Dependovirus in theⅧth report of the ICTV in 2004.Muscovy duck parvovirus, another member of Dependovirus, was identified the pathogen of Muscovy duck three weeks disease (MDTW). It was reported that only Muscovy duck was infected by MDPV and showed symptoms similar to GPV infection in Muscovy duck. The nucleotide sequences homology of GPV and MDPV was 81.9%, so the general serological methods and technique of molecular biology were difficult to distinguish these viruses. Recent years, MDPV became seriously as the scale of feeding goose and Muscovy duck increased rapidly in China. Therefore, it is important to develop a technique to diagnose GPV and MDPV on antigen and antibody in practice.1. PCR method specific for detecting GPV not MDPV was reported. According to Limn's and CHU's reports, primer AL18R2/AL18F2 amplified 806bp fragment of VP3 gene of GPV and primer GPVR/GPVF amplified 2200bp fragment of VP1 gene of GPV. The PCR products DNA by primer AL18R2/AL18F2 were sequenced and compared with GPV B and MDPV GD strains reported in GenBank. The results showed that the nucleotide sequences homology were 97% and 99%. However, the homology between the two PCR products by Limn's primer was only 81.1%. PCR products by primer GPVR/GPVF were compared with GPV B and MDPV GD strains, the nucleotide sequences homology were 97% and 99%. However, the homology between the two sample PCR products by CHU's primer was only 80.3%. It was concluded from these results that the PCR technique using Limn's and CHU's primers may differential diagnosis MDPV and GPV if combined with DNA sequencing, which was named PCRS technique.Disadvantage of the PCRS were found including time-consuming (for eight days) and costly (Y80/sample). To this end, according to the report of Zoltan Z, we designed primer HJU/HJL. PCR results revealed that only GPV reference strain became positive strap, MDPV reference strains was negative. Compared the nucleotide sequences of PCR products with GPV B strain, the homology reached to 99%. The results showed that the PCR primers HJU/HJL can differentiate GPV without depend DNA sequencing. The costs for each sample decreased to Y29 and taken about 30 hours.2. Using the specific PCR, the tissue collected ill goslings were detected. The positive rate of the tissue samples from the 11-day-old Gosling were 75% (Pancreas), 75% (duodenum), 66.7% (kidney) respectively. All of the heart, liver, spleen, lung, kidney, pancreas, brain tissue of 48-day old ill goose were positive, and the duodenum 75% positive. On conclusion: the pancreas, kidney and duodenum contained higher titer of the GPV and were collected firstly if using PCR detection. It was pay attention to 48-day-old geese infected GPV, which showed that the susceptible age of gosling to GPV increased.3.Total of 13 geese samples collected from SiChuan,ChongQing,GuangDong and GuangXi in China were detected by PCR using primers AL18R2/AL18F2 and GPVR/GPVR, 8 and 4 samples became GPV positive. Compared with GPV B strain, the homology of samples' PCR products of 8 positive were 96.20%～98.59% in nucleotide sequence and 98.11%~100% in amino acid sequence induced eight sites, I.e. 3212bp, 3891bp, 3838bp, 3824bp, 3723bp, 3442bp, 3410bp, 3345bp showed more mutation in the PCR products. Amino-acid mutation sites the 463th glycine (G) into serine (S), and 485th threonine (T) into alanine (A). Compared with GPV B strain, the homology of nucleotide sequence of the 2 DNA sequence results of PCR products (2200bp) were 96.0% and 96.3%, and the homology of amino acid sequence were 95.3% and 95.2%. The high mutation domain of PCR products lied in 3899bp～4032bp, which had 12 mutation sites. And the next mutation domain lied in the sequence between VP1 and VP2 initiators, which had 8 mutation sites. Combined with the analysis of hydrophilicity and antigenicity, it was conclusion that GPV strains from the four provinces showed not obvious variation.4. SRW strain, an attenuated GPV by embryonated duck egg passage, was cultured by embryonated duck egg, and the allantoic fluid was harvested from the death embryo. GPV antigen, extracted and purified by Sephadex G-200 after treated with chloroform and precipitated by PEG6000, was used to establish the direct ELISA to detect the antibody in goose serum against the GPV. The optimal coating concentration was determined as 41.75μg/mL. Serum of Rabbit anti-goose IgG and goat-anti-rabbit IgG labeled HRP was dilution to 400 times and 60000 times. The max-level of antibody maybe detected at the 4th or 5th week post firstly inoculated SRW strain attenuated vaccine for 30～50 day-old goslings. The positive results could maintain for 12 weeks.