| Newcastle disease(ND)is caused by the Newcastle disease virus(NDV),which is a highly contagious infectious disease and worldwide epidemic.The NDV-infected poultry and wild birds usually manifest respiratory and neurological symptoms and other clinical manifestations such as diarrhea and depression.Generally,wild birds maintain asymptomatic responses to NDV infection,which leads to a potential risk of spreading the virus.It can cause an ND outbreak in any place where poultry is farming.The genotype of epidemic NDV strains in China is mainly sub-genotypes VI and VII.These viruses have been widely detected in most areas especially in recent years,posing a great threat to the poultry industry.Therefore,continuous monitoring of the epidemic ND and further investigation of epidemiological transmission and evolution dynamics of NDV in poultry are vital for elucidation of the characterization of viral evolution and genetics.It is necessary to provide early warning for the emergence of a new sub-genotype NDV and better prevention and control of ND outbreaks.In this study,the NDV surveillance on the serologic and etiological examination in Shaanxi and Gansu provinces was analyzed during the past six years.An NDV strain(GN-17)was isolated from the Gannan region.The genome of this NDV isolate was sequenced by next-generation sequencing belonging to sub-genotype VII.The virulence determination was identified as a velogenic NDV strain.A q PCR method was developed to detect the viral load using the NDV M gene.The differential pathogenicity of different inoculated routes’ response to this NDV isolate was investigated using the q PCR method.At the same time,the effects of compound probiotics on pathogenicity and regulation of the intestinal immune system in chickens to genotype VII NDV was further analyzed using the transcriptome sequencing technology.Meanwhile,an indirect ELISA assay was established based on the monoclonal antibodies against the GN-17-HN protein.The enhancement effect of compound microecological preparations on immunity was assessed using the ELISA assay.The main results of this study are as follows:1.According to surveillance results of the serological and etiological of Newcastle disease virus in Shaanxi and Gansu regions from 2017 to 2022,it was found that the vaccination effect was unsatisfactory results in Tongchuan,Qingyang,and Gannan regions.Therefore,there is a risk of ND introduction and infection in immunized chicken flocks in these regions.Meanwhile,the phenomenon of NDV infection may exist in some chicken flocks in partial regions of Gannan,Yan’an,Hanzhong,and Baoji.Therefore,it is imperative to expand the scope and conduct in-depth surveillance of Newcastle disease(ND),strengthen the evaluation of the vaccine immunization effect,and advance monitor for the emergence of new NDV genotypes.Greater attention must be paid to ensuring high-quality ND surveillance to prevent outbreaks or continued impacts on chicken populations and breeding environments in live and movement areas,particularly for wild poultry and waterfowl.2.A genotype VII NDV strain(GN-17)was successfully isolated and subjected to genetic evolution and virulence analysis.The isolate exhibited a high homology of 99%with Chicken/China/Liao-ning/03/2008(KC542901.1).The F0 protein cleavage site amino acid sequence was identified as 112 RRQKR ↓ F117.Molecular biology results were consistent with the IVPI,ICPI,and MDT values of GN-17 at 2.88,1.91,and 39.2 h respectively,indicating that the isolate is an immediate type NDV.3.A q PCR method for detecting tissue viral load was established.The pathogenicity and virus proliferation response to the GN-17 isolate infection was detected under different inoculation routes.The results showed that GN-17 infection caused obvious lesions in the spleen,cecum,tonsil and rectum in the infected chicken.At the same time,it was found that the infected chicken had higher morbidity and mortality under the inoculation route of intravenous.Meanwhile,the infected chickens had a slower course of the disease and mild clinical symptoms under the inoculation rote of eye drops and nasal drops,and cloacal.However,the virus enables significant proliferation in different tissues.These above results provide a method for the diagnosis and epidemiological study of ND in poultry production and also provide a new idea for vaccine development and immunization for NDV.4.The pathogenicity of GN-17 in chickens was studied through clinical trials and transcriptome sequencing technology.The complex microecological preparations(e.g.,Bacillus subtilis,Bacillus licheniformis,Lactobacillus,Enterococcus faecalis,Clostridium butyricum,and astragalus polysaccharide)could significantly enhance the immune effect,which could be used as immune-enhancing agents for clinical production,promote the intestinal development of chickens,delay the course of the disease,and reduce the infection damage.The compound microecological agents can promote the production of Ig A in the intestinal immune system and cause gene expression changes in the several related pathways that are involved in the intestinal immune system,such as the NOD-like receptor signaling pathway,RIG-I-like receptor signaling pathway,Toll-like receptor signaling pathway,PPAR signaling pathway,and so on.This study provided the scientific basis and research foundation for effective prevention and control of NDV.5.The monoclonal antibodies were screened and prepared based on the template of the GN-17 HN gene.Subsequently,an ELISA assay was established for antibody-level detection based on the monoclonal antibodies targeting the GN-17 HN gene.The detection efficiency of ELISA was exhibited higher than that of the HI test.The analysis results of the animal experiment suggest that compound microecological agents can serve as immune enhancers and enhance the immune effect of inactivated NDV on chickens.This study presents a significant method for NDV serological detection and confirms the efficacy of compound microecological agents as immune enhancers for NDV,which enable application for production and practice. |
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