Biological Characteristics Of The In Vitro Subculture Strains Of Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia is a highly contagious respiratory disease caused by Actinobacillus pleuropneumoniae Which would cause viral infection of the respiratory tract in pigs and severe economic losses globally in the swine industry.Currently,inactivated vaccination and subunit vaccination are the main measures for preventing and controlling PCP.But the cross-immune protection rate of a variety of serotype strains is low.The live attenuated vaccine may be the way to improve the cross protection between different serotype strains.Culturing APP continuously in vitro obtains the subculture strains.The biological characteristics and genomic characteristics of some subculture strains were studied to screen out the strains with weakened virulence.And analyzing the mutation site of their genes.The study will provide the material of attenuated live vaccine.1.Constructing subculture strains and investigating the virulence with mouse model APPJL01-1 was cultured in vitro at 37°C until the 500th generation,and the subculture strains were named from A37-1 to A37-500.First,different doses of APP JL01-1 were infected with Kunming mice,and the half lethal dose(LD₅₀)of JL01-1mice was determined to be 2.76×10⁶ CFU.Nine subculture strains were selected to infected with Kunming mice with the dose of about 1-2 times of LD₅₀.By comparing the mortality and the amount of bacteria in the lung,the depressed of mortality was found of the isolates after 120 generation.APP JL01-1 was cultured in vitro at 40°C until the 270th generation,and the subculture strains were named from A37-1 to A37-270.Five subculture strains were selected to infected with Kunming mice with the dose of about 1-2 times of LD₅₀.By comparing the mortality,the enhanced of mortality was found of the subculture strains.2.The growth and stress resistance of APP subculture strains in vitroIn order to study the virulence,growth and stress resistance in vitro,A37-70,A37-100,A37-119,A37-120,A37-270,A37-320,A37-430 and A37-500 were used to detect their hemolytic activity,ability of survival in the simulated environment in vivo and the ability of the biofilm to form in the simulated environment in vivo.It was found that the hemolytic activity of the subculture strains was not statistically different from the JL01-1.After 120 generations of subculture strains the growth capacity is enhanced at 37°C,42°C,anaerobic environment and lack of iron.At low p H,growth capacity of all subculture strains was reduced.In addition to 500generation strains,ability of the biofilm formation of other subculture strains are weaken in different environments.3.Comparative genomics analysis between APP JL01-1 and subculture strains Whole genome sequencing of subculture strains 37-120,A37-270,A37-320,A37-430,A37-500,which were depressed mortality and changed in vitro experiments.There was a total of 43 SNP mutations,20 deletion mutations and 35insert mutations.No large fragments shifting were found.The RNA chaperone gene hfq has SNP mutations,deletion mutations gene lpx C＿1 and two-components signal transduction systems gene pho P are involved in bacterial colonization,infection and environmental adaptability,which might be a reason for the decline of subculture strains.The mutations of Cell division association genes,fts Z＿1,fts Q,fts A and cell wall synthesis genes ddl＿1,ddl＿2 might be the reason of faster growing.In addition,mutations in environmental adaptation-related genes hfq and pho P may also be the reason of the changes in biofilm formation and growth.No mutations were found in the apx gene,it is consistent with the detection of hemolytic activity.