Research On The Diagnostic Method To Differentiate Field Infection From Vaccination In Avian Infectious Disease

Avian Influenza (AI) is one of fatal infectious disease caused by influenza A virus in avian. It was first reported in Italy in 1878. Subsequently, different strains were found and caused enormous economic losses to the avian industry throughout the world. At present, highly pathogenic avian influenza (HPAI) is listed in a infectious diseases by World Organization for Animal Health (OIE). Recently, from late 2003 to early 2004, H5N1 HPAIV broke out among poultry throughout Asia, leading to not only tremendous economic losses in poultry industry, but also human infections and deaths. It shocked mankind and the govements of the world. A common viewpoint is that prevention of AI is closely linked to persistent and steady development of avian industry and humankind's health.It is also known that a common practice to vaccinate chickens against ATV and monitor AIV more closely. The wide presence of AIV warrants a diagnostic method to differentiate AIV infected and vaccinated birds. Nonstructural (NS) proteins can be used as markers for infection because viral infections induce antibodies to both structural and nonstructural antigens while immunization with chemically inactivated virus vaccines elicit only antibodies to structural proteins. This dissertation mainly targets establishing a diagnostic method by using non-structural protein NS1 of AIV to detect the antibodies, and differentiating field infection from vaccination in avian disease. The research results are summarized as following:1. The cloning of AIV NS1 gene and it's expression in E.coli.A pair of primers were designed according to the sequence of the nonstructural protein (NS1) of the Influenza virus and a 654 bp gene segment was amplified by RT-PCR with the template of influenza virus (H9N2) isolated. The amplified DNA fragment was cloned into pMD18-T Vector and identified with SalI and XhoI restriction enzyme analysis and sequencing, then inserted into an expression vector (pET-28a) to yield the expression plamid pET-28a-NSl. After induced by IPTG, the NS1 gene was expressed in BL21(DE3). SDS-PAGE analysis showed that the protein was 30kD and the product of expression was specific to antisera against AIV by protein dot blot analysis and Western blotting assay.2. The development of NS1-ELISAThe NS1-enzyme linked immunosorbent assay (NS1-ELISA) based on the recombinant protein which has been purified, denatured, and renatured was developed. The optimal conditions for the ELISA were first established by matrix titration. Theamount of NS1 protein for coating ELISA plates were determined to be 1.75fAg /well. The sera dilution was determined to be 1:40. The cut-off value was established to be 0.369 (mean OD value + 2SD = 0.192+2x 0.088) through examining 94 negative sera samples. To evaluate the specificity and sensitivity of the NSl-ELISA, 126 serum samples were collected from experimentally manipulated chickens. Using HI as the reference methods, the specificity of the NSl-ELISA was 93.1% and its sensitivity was 94.4%.To further evaluate the specificity of the test, the NSl-ELISA was used to test a panel of sera, including sera from chickens with Newcastle disease (ND), infectious bursal disease (IBD), infectious bronchitis (IB), infectious laryngotracheitis (ILT), Marek's disease (MD), fowl adenovirus (FAV), sera from AIV vaccinated chickens, AIV infected chickens and SPF chickens. Only sera from AIV infected chickens was found positive by NSl-ELISA, indicating the NSl-ELISA was highly specific.To test reproducibility of the NSl-ELISA, 6 serum samples with antibody levels, ranging from negative to highly positive were tested repeatedly (6 times) with ELISA plates coated with two batches of NS1 protein. Each serum sample was assayed six times with each batch of coated plates. Data showed there were no significant differences among the six readings for each sample.3. Development and Application of Enzyme-linked Immunosorbent Assay Test Kit to Detect AntibodiesWe developed an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to AIV NS1 for differentiating infected chickens vs. chickens immunized with inactivated avian influenza virus. The kit consists of seven reagents and two pieces of 96 well immunomicrotiter plate. There were high specificity, sensitivity and reproducibility in detecting serum samples with the test kit. It was stable well when storing at below -20 °C for four months up to now. A total of 823 chicken sera samples were sent from several field farms such as Hubei, Shenzhen and Henan. The above results indicated that the NSl-ELISA can be used to differentiate infected vs. immunized chickens. It is a potentially valuable diagnostic tool for AI eradication campaigns and control programs.