| Porcine reproductive and respiratory syndrome virus(PRRSV),an enveloped and single-stranded RNA virus,is the main pathogen causing reproductive dysfunction in sows and respiratory symptoms in swine at various ages.Because of the lax introduction of breeding pigs and the non-standard vaccination,as well as the high variability and frequent recombination of RNA viruses,PRRSV has been prevalent and rampant for more than 20 years after its appearance in China,and two novel genotypes have been detected.Pseudorabies virus(PRV)can not only cause neurological symptoms in piglets,but also cause reproductive disorders in sows,and is often mixed with PRRSV in clinical infection.PRV had been effectively controlled after it appeared in China,but new variant has been found.At present,the domestic epidemic is still unstable,and there is a risk of outbreaks.The prevention and control of these two diseases still lie in vaccination.In this study,we conducted an epidemiological survey on the samples collected from some pig farms in China during 2017 ～ 2019,and conducted an isolation,identification,and virulence study based on the screened PRRSV.Then we prepared a bivalent inactivated vaccine with the g E gene deletion virus of the PRV variant,and conducted quality inspection,safety and efficacy evaluation of the bivalent inactivated vaccine,to apply it to disease prevention,control,and purification.1.Molecular epidemiological investigation of PRRSV in some regions of China and the isolation and pathogenicity identification of a highly pathogenic strainThe epidemiological investigation of PRRSV was conducted by using two national standard RT-PCR methods on 1441 samples collected from pig farms of different scales in14 provinces and cities in China from 2017 to 2019.Based on these samples,the epidemic strains were screened for virus isolation,and the isolated one was further identified through electron microscopy test,indirect immunofluorescence test,sequence analysis,and the virulence was verified by animal regression tests.The results of the epidemiological investigation showed that the positive rate of collected samples was 22.14%,and the HPPRRSV infected samples accounted for about 52.98% among these positive samples,the positive rate is 11.73%.The virus particles with a diameter of 50 ～ 72 nm were observed by electron microscopy after preliminary purification of the virus suspension.Specific green fluorescence was observed by fluorescence microscope after indirect immunofluorescence staining.The sequence analysis of nsp2 gene and ORF5 gene showed that the two genes of the isolated strain belong to the HP PRRSV strain,which is the closest to the TJ strain.Regression test results showed that the daily weight gain of 5-week-old piglets after being infected with the isolate was significantly reduced,the body temperature increased,and severe clinical symptoms such as coughing,body tremor,and paralysis of hind limbs appeared.Histopathological results showed that the lungs of the pigs showed typical symptoms of diffuse interstitial pneumonia caused by PRRSV infection.In addition,one piglet died,with a mortality rate of 20%.Thus,it was named RP19,which lays a foundation for further research.2.Preparation and safety evaluation of the PRRSV、PRV bivalent inactivated vaccineSince PRRSV infection in pigs will lead to immune suppression,it is often clinically mixed with other pathogens,and PRV is one of the common pathogens.To prevent and purify PRRSV and PRV,the isolated prevalent strain PRRSV-RP19 and the g E knockout PRVΔg ERP15 were used as antigens,and the cultured virus solutions were concentrated and inactivated separately,which were equally mixed then were used for the preparation of a bivalent inactivated vaccine and two single inactivated vaccines according to the water-oil ratio of 1:2.The quality inspection and safety evaluation of the vaccine were carried out.The results of quality inspection showed that the indicators of the three vaccines were qualified.The results of safety evaluation showed that there were no clinical signs except for transient fever in the pigs after being immunized with 4.0 m L/each dose of the bivalent vaccine.The general observation of organs after autopsy was normal without lesions,and the pathological histology was normal without lesions.3.Efficacy evaluation of the PRRSV、PRV bivalent inactivated vaccineIn order to applicate the PRRSV、PRV bivalent inactivated vaccine into clinics,two trial-produced single vaccines and one bivalent inactivated vaccine were vaccinated into 5-week-old piglets,and a challenge protection test was performed 28 days later.The results showed that some piglets in PRRSV single vaccine group developed mild symptoms after challenged,and there was no difference in weight gain and no serious pathological changes in organs,and the protection rate reached 60%.In PRV single vaccine group,piglets have no change in body temperature,no difference in weight gain,no obvious pathological changes in organs,and the protection rate reached 100%.The bivalent vaccine group was challenged with PRRSV and PRV separately after immunization.Some piglets in the PRRSV group developed mild symptoms of viral infection.The weight gain was significantly different from that of the control group.There was no serious organ disease and the protection rate reached40%.The body temperature of one piglet in the PRV group increased temporarily,and there is no difference in weight gain,no obvious organ lesions,and the protection rate is 80%.The results indicated that the trial-produced bivalent inactivated vaccine has an excellent protective effect on PRV,but an indifferent protective effect on PRRSV. |
PDF Full Text Request