| Newcastle disease(ND),caused by virulent Newcastle disease virus(NDV),is an acute,febrile and highly contagious avian infectious disease.Virulent NDV can induce high morbidity and mortality in chickens,causing serious economic losses to the poultry industry around the world.ND is classified as a class of epidemic disease in the national medium-and long-term animal disease prevention and control plan of our country.South China,with a humid climate and rich natural vegetation,and there are a large number of live bird markets(LBMs)and a number of migratory bird migration routes,provides favorable conditions for the spread of NDV.This study conducted an epidemiological survey of NDV in Guangdong Province and surveillance of NDV in healthy poultry flocks and environment in Guangdong and Guangxi Provinces from 2017 to 2019.In the epidemiological investigation of NDV,four NDV were isolated by conventional virus isolation and identification methods,all of which were Class Ⅱ NDV.The total positive isolation rate was 0.19%,of which two strains were Class Ⅱ genotype Ⅸ,and the other two strains were genotype Ⅱ.The statistical results show that from the analysis of host distribution,the positive isolation rate of NDV in waterfowl is the highest as high as0.83%,and 0.31% in poultry.From the source distribution analysis,the positive isolation rate of NDV in zoo samples was the highest,up to 1.16%,and 0.26% in poultry farms.During the monitoring of NDV in healthy poultry flocks and environment in Guangdong and Guangxi Provinces,80 NDV were identified from the chicken embryo allantoic fluid of86 HA positive samples,of which 77 NDV were clustered into Class Ⅰ subgenotype 1b,accounting for 96.25% of the positive samples,and the positive isolation rate was 5.02%.The other three NDV belong to Class Ⅱ genotype Ⅱ,accounted for 3.75% of the positive samples,and the positive isolation rate was 0.20%.According to the investigation and surveillance results of this study,it was found that the lentogenic strain of Newcastle disease was prevalent in poultry flocks at present.Conventional detection methods are time-consuming and less sensitive,and it is difficult to carry out high-throughput detection.In this study,through the multi-sequence alignment analysis of NDV,it was found that there was a difference in the number of bases in the open reading frame of Phosphate gene of Class Ⅰ and Class Ⅱ NDV.A pair of primers NDV P1-F and NDV P1-R were designed in the upstream and downstream of the relatively conserved region of Phosphate gene.Using HRM analysis technique,a high resolution melting curve detection method(PCR-HRM)was successfully established for the first time to distinguish between Class Ⅰ and Class Ⅱ NDV.The HRM detection method has the advantages of high specificity,high sensitivity and good stability,and is unable to amplify other avian virus genes that are not NDV.The lowest detection line is 22.5 copies/μL(ClassⅠ)and 26.2 copies/μL(Class Ⅱ).The intra-batch difference of sample melting temperature is 0.00℃～0.13℃,and the inter-batch difference is 0.03℃～0.28℃.Compared with conventional nucleic acid electrophoresis,PCR-HRM greatly reduces the risk of cross-contamination,takes a short time,is easy to operate,and can detect 384 samples at the same time.Clinical samples can be identified according to the melting temperature range of Class Ⅰ and Class Ⅱ NDV.To sum up,through the epidemiological investigation of NDV in Guangdong and Guangxi Provinces,we learned about the prevalence of NDV in farms,live poultry markets and wild birds,and then established a high-resolution melting curve detection method to distinguish Class Ⅰ and Class Ⅱ NDV,and provided a detection method for rapid identification of two types of NDV in clinical samples. |
PDF Full Text Request