Typing,Distribution And Application Of Regional Precision Control Of Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) In Liaoning Region

Porcine Reproductive and Respiratory Syndrome(PRRS)is caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),and the main clinical manifestations include reproductive disorders of sows and dyspnea of piglets,which can cause the death of infected piglets.The mortality rate of suckling piglets is higher than 60%,and that of nursery piglets ranges from 10%to 30%.Highly pathogenic Porcine Reproductive and Respiratory Syndrome(HP-PRRS)can significantly increase the morbidity and mortality with the morbidity of piglets at 100%and the mortality higher than 70%.And the morbidity and mortality of adult pigs can both reach higher than 50%,and sow abortion rate can reach higher than 30%.In pig herds,both classic strains and highly pathogenic strains are prevalent,and these strains need to be clarified and distinguished during daily immunization and diagnosis..In the view of the fact that there is no effective treatment for PRRS,immunization is mainly used to prevent the disease clinically.Strict implementation of immunization is one of the most active and effective measures to prevent and control the disease.At present,the disease is widespread and prevalent in the world.The PRRS epidemic not only caused huge economic losses in the pig industry,but also high risk to human food safety.The occurrence and prevalence of PRRS in Liaoning were studied by using questionnaire survey,etiology,molecular biology and serological methods.The results showed that the dominant serotypes of PRRSV and the occurrence of PRRS was different in various regions of Liaoning,so it was imperative to establish targeted detection methods and precise control strategies according according to the local dominant strains and epidemic trends.At the same time,In order to improve the PRRSV detection system in Liaoning Province,the typing method of PRRSV was established in this study to type the dominant strains of PRRSV prevalent in different areas of Liaoning Province,providing a basis for the formulation and implementatio n of precise immunization and control measures.The predominant serotypes of PRRSV in various regions of our country were different.The occurrence and prevalence of PRRS in Liaoning were studied by using questionnaire survey,etiology,molecular biology and serological methods.The results showed that the dominant serotypes of PRRSV and the occurrence of PRRS was different in various regions of Liaoning,so it was imperative to establish targeted regional accurate typing methods and precise control strategies according to the local dominant strains and epidemic trends.By combining the typing methods established in this study,serology,molecular biology and other methods,a study was conducted on the PRRSV pathogen infection and immune antibodies in the pig herd in the whole province.The precise prevention strategies was recommended and implemented,and finally the immunization effect was evaluated.These provide scientific basis and technical support for the formulation of precise prevention and control measures of PRRS and the guidance of clinical diagnosis and production management in pig farms in Liaoning.The main research contents and results in this study are presented as follows:(1)Establishment and application of PRRSV typing detection methodIn this study,two specific primers were designed according to the two ends of the missing gene in the NSP2 coding region of the PRRSV genome,and the RT-PCR method was successfully established to detect and distinguish the classic strains of PRRSV and the highly pathogenic strains,while the primer concentration,reaction time and reaction temperature were also optimized.The results of the study found that the optimal annealing temperature for this RT-PCR reaction was58?,and the optimal primer concentration in the system was 0.4?mol/L.The results of the specificity test showed that there was no cross-reaction with common swine-infected viruses such as classical swine fever virus,pseudorabies virus,porcine circovirus type 2 and porcine epidemic diarrhea virus in this method,which suggested that this method can provide a good specificity.The sensitivity test showed that this method could amplify the classical strains at a dilution of 10^-3 and the highly pathogenic strains at a dilution of 10^-4.The results of tepeatability test showed that there was no obvious difference among the test results with a good repeatability.The genotyping detection method established in this study provided a higher detection rate for HP-PRRSV,and the detection rate of classical strains was consistent with the detection rate of the national standard detection.This method was suitable for veterinary laboratories and farms to identify the PRRSV,and guided the formulation and implementation of precise prevention and control strategies for PRRSV in Liaoning region,and also provided a fast and effective detection method for early warning of the outbreak of PRRSV.(2)PRRS incidence and pathogen nucleic acid detection and analysis1)The incidence and analysis of PRRS in clinicThrough the investigation of PRRS in 596 pig farms of different scales in 14 prefectures and cities in Liaoning Province from 2014 to 2015,the survey results showed that the overall PRRS clinical incidence rate of pig herds was 7.05%during these two years,and the incidence of PRRS showed a downward trend with the increase of the farming scale;the clinical incidence of PRRS in2014 and 2015 was relatively stable;the lowest incidence of PRRS at 5.06%was found in southern Liaoning in 2015,while the highest incidence of PRRS at 8.96%was detected in northern Liaoning in 2014.The clinical incidence of PRRS presented a relatively popular situation in Liaoning without obvious geographical difference.Therefore,it was essential to operate a better PRRSV immune control.2)Pathogen detection and analysisThe average positive rate of HP-PRRSV pathogen in a hypothetical healthy pig population in Liaoning Province during 2014 to 2018 was 5.96%.In these years,the average positive rate of HP-PRRSV in 2015 was the highest(14.70%)and the lowest was found in 2017(0.97%);for the average positive rate in four quarters,the highest was in the first quarter(8.93%)and the lowest in the third quarter(8.40%);for different areas,the average positive rate in the northern Liaoning region was the highest(7.54%)and the lowest in the southern Liaoning region(4.26%).From 2017to 2018,compared with 2014 to 2016,the detection rate of HP-PRRSV pathogen was decreased in Liaoning Province.Using the PRRSV typing test,the results showed that the number of suspected cases infected with highly pathogenic strains in 2016 and 2017 was higher than that of cases infected with classical strains.And the number of cases infected with classical strains in 2018 was higher than that infected with highly pathogenic strains.In general,the highly pathogenic strains were the main epidemic strains,accompanied by the epidemic of classic strains in Liaoning Province.In recent years,the prevalence of highly pathogenic strains has declined,and the presence of classic strains has increased accordingly.When formulating immunization policies,attenuated vaccines should be selected according to the specific conditions.The results of nucleic acid detection of major infectious diseases in pigs showed that the mixed infection rate of PRRSV and PCV2 was the highest(43.72%),and the lowest mixed infection rate was with PRV(0.64%).3)Molecular evolution of PRRSV and other major viral diseasesThe gene sequences determined in this study were all HP-PRRSV strains.The homology of theses ORF5 genes was 91.5%?99.0%,and the homology of these NSP2 genes was 92.9%?99.9%.Compared with the amino acid sequence of VR-2332 strain,the 39^th position of the ORF5amino acid sequence mutated from L to I,which was same as the highly pathogenic strains including JX A1,HUN4 and TJM;And also,compared with the amino acid sequence of VR-2332,the 13^th position mutated from Q to R,and the 151^th position of LNeast2015 mutated from R to K;Meanwhile,the aa 137^thposition of all stains was S,indicating that these strains identified in this study were all wild strains.The evolutionary tree analysis showed that a certain degree of genetic variation has occurred in the PRRSV strains in Liaoning Province,and these strains were closely related to vaccine viruses.The appearance of these vaccine strain-related isolates might be related to the widespread use of attenuated modified active PRRS vaccines.Combined with amino acid sequence analysis,these results indicated that these strains might be genetic recombinant strains of wild and vaccine strains.The sequence analysis of the target genes of CSFV,PRV and PCV2 showed that the pathogens had varying degrees of variation and some highly pathogenic strains were found.Some pathogens in Liaoning were closely related to the pathogens in other regions,which means there was higher risk of introduction.(3)Detection and analysis of antibody after immunization of PRRSV vaccineThe average positive rate of PRRSV antibodies in pig populations in Liaoning Province between 2014 and 2018 was 91.33%,of which the highest rate was 94.26%in 2015 and the lowest rate was 87.76%in 2017.For the average positive rate of antibodies in four quarters,the highest rate was found in the fourth quarter(93.31%)and the lowest one in the first quarter(89.99%).And for the regions,the average positive rate of antibodies in the eastern Liaoning region was the highest(93.82%)and the lowest was found in the southern Liaoning region(4.26%).The positive rate of PRRSV antibody was always above 70%,which can provide immune protection to the population.The total positive rate of PRRSV antibody in swine serum samples at different growth stages was 94.39%,with the lowest in the weaned piglets(81.67%),and the highest in gilts,sows and boars(100%);the overall positive rate of pathogens was 6.52%,of wh ich the lowest rate was found in boars(0%)and the highest rate in fattening pigs(16.67%);among the serum samples of different breed pigs,the positive rate of PRRSV antibody in the large white herd was the highest(96%),and the binary hybridization had the lowest rate(78%).In total,the positive rate of PRRSV antibody at different ages and from different breeds all could reach more than 70%,and there was no significant difference among these samples.The results of dynamic antibody detection of piglets immunized with attenuated vaccine showed that the antibody level in piglets was low in 4 weeks after immunization,and it began to rise at the 5^th week after immunization,and the antibody level reached its peak at 8?9 weeks and could last until 16 weeks after immunization.Piglets immunized with the highly pathogenic strain of PRRSV attenuated vaccine reached the peak of antibody positive rate one week earlier than those immunized with the classic strain of PRRSV attenuated vaccine,but there was no significant difference between these two kinds of vaccine.Therefore,it is recommended that piglets should be immunized at age of 25 to 35 days for the first time,and 4 months later for the second time.(4)The implementing effect and analysis of PRRS precise immunization control measuresIn combination the established PRRSV typing detection method with immunization procedure in this study,the precise immunization prevention and control strategy was recomme nded,which achieved good results after implementation in pig farms,and had a high reference value for the formulation of PRRSV prevention and control plan and immunization control measures i n pig farms in Liaoning region.After the implementation of precise immunization prevention and control measures in the testing farms,the blocking rate of immune antibodies in pigs was significantly increased and the antibody level tended to be consistent,most of which reached higher than 0.8.PRRSV pathogen nucleic acid test results showed that the detection rate of pathogens after immunization was significantly reduced.Comparing the production indexes of sows and suckling piglets before and after immunization,the incidence of abortion,mummy fetal and total mortality of sows after immunization were significantly reduced,whilethe fetal retention rate and piglet survival rate were increased.The herds immunized according to the recommended immunization procedures produced lower antibody dispersion,less variation in antibody levels and better overall protection.The investigation and testing results of the sampled pig farms in Liaoning showed that all pigs were immunized with highly pathogenic strains of PRRSV vaccine in 2016.In 2017 and 2018,less highly pathogenic strains of PRRSV vaccine was used,while more classic strains of PRRSV vaccine was used.The incidence of PRRS decreased after the application of precis e immunization measures in western and northern Liaoning.Compared with 2017,the number of clinical cases detected with highly pathogenic strains of PRRSV was decreased in all regions in 2018,and the number of clinical cases detected with classical strains in western and northern Liaoning was decreased.Compared with 2017,the antibody positive rate in we stern and northern Liaoning increased in 2018.Compared with the farms without precise immunization measures,the farms with precision immunization prevention and control measures in 2018 had lower pathogen positive rate,lower average incidence,and higher antibody positive rate.When formulating and implementing immunization prevention and control strategies,the pig farmer managers should select vaccines and immunization procedures suitable for the farm based on the PRRSV trends on and surrounding the farm,as well as in the entire county and the city,and even the province.If there was no HP-PRRSV epidemic in the surrounding fields and counties,PRRSV negative field should try to select classic strains attenuated vaccines for immunization;PRRSV-positive farms should first determine which PRRSV serotype infection occurred on the farm,and then selected the attenuated vaccine for emergency group immuniza tion.