Establishment And Application Of Detection Methods For Avian Metapneumovirus

Avian metapneumovirus was first described in South Africa, which mainly causes an upper respiratory tract or central nervous system diseases in turkeys and chicken, such as Turkey rhinotracheitis and Swollen head syndrome. Now, apart from Australasia all major poultry raising regions of the world have reported the presence of aMPV. Avian metapneumoviruses have been further classified into four subtypes (A, B, C, D) , subtypes A and B are reported all over the world, subtypes C and D are respectively reported in the United States and France.In 1999, China had successfully completed separated and reported this virus for the first time thus proved the epidemic of aMPV in our country. Serology investigation showed that the infection of aMPV can reach 100% in breeding bird in Shandong Province. Subtype A was detected in 39% positive from chicken samples by RT-PCR method in 2008, but subtype B was negative. In order to further understand the prevalence of aMPV, this research focused on etiology and serologic research for the disease, and then it provides the theory basis of prevention and control.1. Establishment and application RT-PCR for detection of aMPV.According to the genomic sequences of F gene of aMPV published in Genbank, a pair of primers was designed for amplifying the 725 bp fragment in RT-PCR experiments. The specificity test showed that the RNA of aMPV could specially amplify the 725bp fragment, but other nucleotide extracted from H9N2, NDV and IBV were negative. The sensitivity test showed that as low as 1.45 pg/μL of aMPV's cDNA could be detected by RT-PCR method. Then 492 samples of broilers with signs of respiratory disease were detected and the positive rate was 43.09%. We randomly picked 11 positive samples from different areas for further confirmation. Based on the analysis we could confirm that all the positive samples belonged to subtype B.2. Establishment and application nucleic acid probe for detection of aMPV.The RT-PCR product of the 725 bp which was recovered and purified was labeled with DIG as cDNA probe for detection of subtype B aMPV. The hybridization assay of specificity showed that the RNA of aMPV displayed specific positive reaction, but other nucleotide extracted from H9N2, NDV, IBV, E.coil and the tissues of SPF were negative. The sensitivity test showed that as low as 5 pg of aMPV's DNA could be detected by DIG-labeled probe. The samples of 605 broilers and 122 ducks were collected from ?ocks in Shandong Province, and then dot blot hybridization was used to detect aMPV, the results showed that the detection rates of the two kinds of brids were 36.59% and 34.51%, respectively. So the probe could be used in the detection of aMPV. This investigation also showed that aMPV was present in China and the infection was common in broilers and ducks on some farms in Shandong Province.3. Establishment and application of Indirect ELISA for detection of aMPV antibody.The purified and recombinant protein was used to coat the 96 pores ELISA board as antigen, which was used to coat a 96 well high affinity ELISA plate as antigen. A series trials were performed for the groped optimal reaction conditions of the indirect ELISA and the results were obtained as follow: the best coating quantity of antigen was 800ng per hole; Serum was diluted 50 times; The best coating conditions of recombinant protein was 4℃overnight and the best coating liquid was carbonate buffer solution (liquid pH9.6); The best block solution was 5% skimmed milk powder, the best block time was 1h at 37℃; The best reaction time of serum was 90min; The best diluted time of enzyme-linked secondary antibodies was 1000 for reaction 60min; the best reaction time of substrate was 10min; the cut-off value of positive result was 0.292 (Cp = X + 3SD), the negative result was 0.258 (Cn = X + 2SD). Moreover, the established indirect ELISA method with the advantages of good repeatability and specificity offered a foundation to aMPV diagnostic kit. 1139 Chicken serums from seven regions of Shandong were detected by this method. The results showed that the general positive rate was 37.05%( 422/ 1139 ), including the lowest 11.41%(42/368) of commercial egg-chickenyer and the highest 62.95%(226/359) of broiler breed .The positive rate of commercial broiler was 37.38%. The results showed that aMPV was widespread in chickens of certain parts in Shandong province.