Molecular analysis of APV and PBFDV and the development of a sandwich ELISA for the detection of APV infections in psittacine birds

• Main Authors: Chih-Ming Hsu, 許志明
• Other Authors: 蔡向榮
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/21180018918731432827

碩士 === 國立臺灣大學 === 獸醫學研究所 === 94 === Avian polyomavirus (APV) infection and psittacine beak and feather disease (PBFD) are the most common viral diseases of psittacine birds. APV was first isolated from budgerigars in the early 1980s. APV belongs to genes Avipolyomavirus of the family Papovaviridae and causes acute fatal disease in young budgerigars with 100% mortality rate. APV infection is also known as budgerigar fledgling disease. PBFDV, categorized into genes Circovirus of the family Circoviridae, affects over 60 species of wild and captive psittacine birds. In this study polymerase chain reaction (PCR) was applied to diagnosis either APV or PBFDV infections in psittacine birds of Taiwan. From 2002 to 2005, the positive rate of APV, PBFDV, and APV/PBFDV infection over 165 cases were 15.2%, 41.2%, and 10.3% respectively. APVs indicated over 97% nucleotide identity in VP1 and T antigen coding regions. PBFDVs had over 92.2% and 83.3% nucleotide identity in ORF V1 and ORF C1 sequences. Another aim of this study is to prepare Sandwich ELISA for detection of APV and to analyze the specificity and sensitivity of Sandwich ELISA. First of all, we applied prokaryotic (E. coli.) system to express the viral structural protein VP1. The expressed VP1 were specifically recognized by his-tag and anti-APV antibodies. The recombinant protein was used as an immunogen to inject BALB/c mice for preparation of hybridomas which produced anti-VP1 antibodies. Eight monoclonal antibodies secreting hybridomas were obtained as determined by ELISA and Western blot. After screening of these 8 monoclonal antibodies, one monoclonal antibody, D10-6, is used to develop the Sandwich ELISA for the detection of APV. No significant difference was formed between PCR and Sandwich ELISA at the sensitivity. The Sandwich ELISA could be applied for diagnosis of APV infected budgerigars, and could further be applied to immunoassay strip for the rapid diagnosis of APV infection in budgerigars.