Isolation And Identification Of Pigeon Paramyxovirus Type 1 And Preparation Of The Inactivated Vaccine And Egg Yolk Antibody

Pigeon paramyxoviruses type 1（PPMV-1）,commonly known as pigeon Newcastle disease,is one of the most important pathogens in pigeon breeding.PPMV-1 is a mutant antigenic strain of Newcastle disease virus（NDV）in pigeons,and the most common PPMV-1 in China belongs to type VI of NDV gene.In recent years,the outbreak of Newcastle disease of pigeon has occurred in many provinces of China,the morbidity and mortality of infected pigeons are high,causing great losses to the pigeon breeding industry.Vaccination is the most effective means for the prevention and control of viral diseases.However,there is no commercial vaccine for the prevention and control of pigeon Newcastle disease in China at present.Chicken Newcastle disease vaccine is mostly used clinically.It is urgent to develop biological agents specifically for Newcastle disease.In this study,PPMV-1 strain was isolated from clinically infected pigeon samples,and genetic evolution analysis was conducted.Inactivated vaccine and egg yolk antibody were further prepared using the isolated PPMV-1 strain,respectively,to determine their immune protection effect and treatment effect,in order to provide materials for the prevention and control of pigeon Newcastle disease.The study yielded the following results.1.A pigeon paramyxovirus type Ⅰ was successfully isolatedIn 2019,PPMV-1 occurred in a public house in Ningxia.The infected pigeons showed twisted head and neck shape,and some of the infected pigeons breathed with purring and excreted green and loose stools.Autopsy showed that the lungs were congested with bleeding points;the liver is swollen with hemorrhagic spots;kidney enlargement;Ingluvies hemorrhage;there are hemorrhagic spots below the cuticle of myogastric;intestinal mucosa edema.Microscopic examination showed that lung congestion,alveolar wall skin cells exfoliated,there were red blood cells in the alveolar cavity;hepatic cord disorder,hepatocyte enlargement;the epithelial cells of renal tubules were degenerated and necrotic,and there were a few red blood cells in renal interstitium.The trabecular structure of the spleen was not clear,the number of lymphocytes decreased,and there were red blood cells between the tissues.PPMV-1 strain was isolated and identified as Pigeon/NX by RT-PCR and sequencing after blind transmission of SPF chicken embryos for 3 generations.The MDT and ICPI of Pigeon/NX were 96 h and 0.41,respectively.The whole genome sequence of Pigeon/NX was 15192 nt,and the sequence of genome was3’-NP-P-M-F-HN-L-5’,which was consistent with NDV genome characteristics.Phylogenetic analysis showed that Pigeon/NX was closely related to pi/CH/LHLJ/131237and pigeon/China/BJ/2013,but was far related to La Sota and B1 strains commonly used in the market.The cross hemagglutinant inhibition test was performed using both Pigeon/NX and La Sota,and the R coefficient was 0.71（less than 0.8）,indicating that there were antigenic differences between Pigeon/NX and La Sota strains.Phylogenetic analysis of Pigeon/NX F gene showed that Pigeon/NX was in the same branch as VIb subtype NDV of Class II gene.The amino acid sequence at the cleavage site of Pigeon/NX F protein was¹¹²R-R-Q-K-R-F¹¹⁷,which was consistent with the cleavage site of other PPMV-1 and avian NDV virulent strains.2.The inactivated Pigeon Newcastle disease vaccine was prepared from isolated Pigeon/NX and its challenge protection test was carried outPigeon/NX was amplified by SPF chicken embryo and EID₅₀ was 10^-7.33/0.2 m L.The oil emulsion inactivated vaccine was prepared by collecting Pigeon/NX alluvial fluid with HA titer greater than 8（log2）.The preparation of Pigeon/NX inactivated vaccine was oil-in-water type.No bacterial growth was observed in LB and TSA medium,and the viscosity test result was 5.2 s.Pigeon populations were inoculated with 2×10⁶ EID₅₀ of prepared Pigeon/NX vaccine and commercial La Sota inactivated vaccine,respectively.After 14 days,the same dose was used for enhanced immunization.Serum antibody levels were detected every 7 days after the first immunization.The HI titer of antibody was0.25±0.25（log2）.The HI titer of Pigeon/NX antibody was 1.00±0.67（log2）,which was significantly lower than 1.75±0.25（log2）in La Sota group（P<0.05）.The HI titer of the La Sota group was 5.25±0.33（log2）and that of the Pigeon/NX group was 5.00±0.92（log2）at 21 days of first immunity,but the difference was not significant.The HI titer was6.5±0.33（log2）in the La Sota group and 6.25±0.25（log2）in the Pigeon/NX group at 28days of first immunity,showing no significant difference.At 28 d of immunization,the control group,La Sota group and Pigeon/NX group received 5×10⁶ EID₅₀ Pigeon/NX nasal drops.On the second day of challenge,one control pigeon died on the 11th day,two died on the 13th day,and one died on the 13th day in La Sota group.No obvious abnormality was observed in Pigeon/NX group.3.The yolk antibody of Pigeon Newcastle disease was prepared from isolated Pigeon/NX and its therapeutic effect was testedLaying hens were immunized with Pigeon/NX vaccine three times,and eggs were collected every 7 days after the first immunization 14 days.The HI titer of egg yolk antibody was higher than 10.40±0.93（log2）on day 35,which was significantly higher than that on day 21（P<0.01）.There was no significant difference between day 35 and day 70（P>0.05）.Egg yolk antibody was collected from day 35.The prepared yolk antibody was inoculated on LB and TSA medium without bacterial growth.The neutralization titer of the prepared egg yolk antibody（HI titer 11（log2））against PPMV-1 was determined by chicken embryo neutralization test.The results showed that the neutralization titer of prepared egg yolk antibody against PPMV-1 was 14.15（log2）.The serum HI titer of 1 m L yolk antibody（HI titer 11（log2））was 6.25±0.25（log2）at day 1 after subcutaneous injection of squab,and 4.75±0.67（log2）at day 14.Compared with day 1,the serum HI titer was significantly decreased（P<0.01）.After 2×10⁷ EID₅₀ Pigeon/NX infection for 1 day,the yolk antibody treatment group was subcutaneously injected with yolk antibody（HI titer11（log2））1 m L,and observed continuously for 14 days.All the yolk antibody treatment group survived,and all the pigeons treated with PBS died.RT-PCR results showed that on day 5,all the pigeons in the PBS treatment group were positive for PPMV-1,and one pigeon in the yolk antibody treatment group was positive for PPMV-1.On day 7,all the 3surviving pigeons in the PBS treatment group were positive for PPMV-1,and all the surviving pigeons in the yolk antibody treatment group were negative for PPMV-1.On day14,PPMV-1 was not detected in the yolk antibody treatment group.In this study,a strain of PPMV-1 was isolated successfully,and Pigeon Newcastle disease inactivated vaccine and egg yolk antibody were prepared from isolated Pigeon/NX.The results showed that both Pigeon/NX inactivated vaccine and egg yolk antibody had good immune protection and therapeutic effect.These results provide theoretical basis and candidate materials for further prevention and control of pigeon Newcastle disease.