| Waterfowl breeding industry is a characteristic industry in China.The breeding quantity of waterfowl in China ranks first in the world,and the annual output is about 6 billion,accounting for about 80% of the world.The strong consumer demand for waterfowl meat and eggs has accelerated the production of waterfowl breeding industry and made the waterfowl industry develop rapidly.With a good breeding tradition and excellent climate environment in Shandong province,the scale of waterfowl breeding industry ranked first in China,and has achieved rapid industrialization development.The Liaocheng area has a well-developed water system and is the main distribution center for waterfowl breeding in Shandong Province.Based on the investigation and statistics of waterfowl samples in Shandong,the frequent occurrence of waterfowl virus diseases in large-scale breeding farms is an important factor restricting waterfowl breeding industry in recent years.According to the investigation of existing virus samples,the main pathogens that harm waterfowl industry blight in Liaocheng area are Avian influenza virus(AIV),Avian Tambusu virus(ATMUV)and Newcastle disease virus(Newcastle disease virus).NDV),Duck hepatitis A virus(DHAV),Avian Astrovirus(AAst V),Duck parvovirus(DPV),etc.The existing research found that the infection rate of weak NDV was high in this area,which is speculated to be caused by the widespread vaccination of NDV vaccine.Therefore,it is difficult to distinguish between vaccine and wild virus infection through the detection of immunized birds.F protein of NDV is one of the main proteins that determine the virulence of the virus.Based on the characteristics of F gene,a specific reverse transcription quantitative PCR method was established to detect the strong virulence specificity of NDV.In addition,DPV infection causes growth inhibition,resulting in a high elimination rate of stiff ducks and causing huge economic losses to the breeding industry.Based on these reasons,the whole genome sequencing of waterfowl parvovirus is carried out to clarify its genetic evolution,which can speed up the process of identification of DPV strains.The above research enriched the molecular epidemiological investigation data of waterfowl viral diseases,provided data support for the early warning of viral diseases,and provided theoretical basis for prevention,control and purification.The research contents are as follows:1.Epidemiological investigation of important virus in waterfowlIn order to understand the epidemic situation of six virus infections in Liaocheng area,540 diseased materials were collected from major large-scale farms for epidemiological investigation during the period of 2018-2022.The results showed that AIV,ATMUV,NDV,DHAV,AAst V and DPV were 31.67%,14.63%,14.44%,10.74%,4.44% and 2.96% respectively.Single virus infection is dominant,and the typing results of AIV show that H9 with low pathogenicity is still the most popular type.Mixed virus infection accounts for a considerable proportion,and Newcastle disease-influenza double infection accounted for the highest proportion.In Addition,the virus infection has obvious seasonal characteristics,mainly in winter and spring.2.Establishment of RT-qPCR detection method for NDVThe virulence of NDV is closely related to the amino acid sequence at the cleavage site of F protein,so the difference in amino acid sequence has become the target to distinguish strong NDV from weak NDV at the gene level.Based on the genomic characteristics of F gene,a RT-qPCR detection method for NDV was constructed.In the optimized reaction system,annealing temperature of 55℃ and primer concentration of 10 μM showed the best amplification efficiency and single dissolution curve.The detection limit of this method was 1 copy /μL,and it could specifically amplify the virulence,has no amplification to the attenuated strain and other strains.3.Isolation,identification and whole gene sequencing of parvovirusBy separating and identifying the positive samples of parvovirus,5 strains of waterfowl parvovirus were obtained,which named SD187,SD271,SD272,SD274 and SD278.The whole genome of 5 strains was sequenced.The results showed that adenine A content was the highest in its nucleotide composition.In addition,there were three potential glycosylation sites in NS protein and VP protein,and two 14 bp deletion segments in ITR.The Genetic evolution,nucleotide and amino acid homology analysis showed that all the five strains belonged to the DPV branch and were highly homologous.In summary,AIV,ATMUV,NDV,DHAV,AAst V and DPV are common in Liaocheng farms with certain seasonal rules,which is an important reason for the complexity of disease prevention and control in large-scale waterfowl farms in Shandong.A rapid and low-cost RT-qPCR detection method for NDV was established,which can only specifically amplify the virus and detect 1copy/u LRNA concentration.In addition,5 DPV strains were successfully isolated and identified,and the whole genome sequence analysis confirmed that they had more than 99% homology,and were the main DPV endemic strains in Shandong Province.Based on the investigation of waterfowl samples collected in Liaocheng area,this study have understand the epidemic trend of six waterfowl virus infections in Liaocheng area,and then providing data reference for the investigation of waterfowl virus infection in Shandong province and even the whole country and theoretical guidance for the construction of waterfowl disease prevention and control system.By constructing RT-qPCR detection method for virulent NDV,the infection of virulent NDV strains can be distinguished,and vaccine interference can be eliminated,which can provide early warning information for NDV detection and effectively control the occurrence and spread of virulent NDV infection.By sequencing the whole genome of waterfowl parvovirus,the genetic evolution characteristics of detoxified strains were analyzed,which provided technical support for the research and development of waterfowl parvovirus vaccine,effective prevention and control of parvovirus disease and purification,and thus provided technical support for the healthy and sustainable development of waterfowl breeding industry. |
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