Isolation,Identification And Pathogenicity Of Actinobacillus Pleuropneumoniae SDHG-1

Porcine infectious pleuropneumonia(PCP)is a highly contagious and fatal respiratory infectious disease caused by Actinobacillus pleuropneumoniae(APP).Pigs of all ages and genders are susceptible to this disease,which are mainly characterized by acute hemorrhagic fibrinous pneumonia and chronic fibrinous necrotizing pleurisy.PCP caused by APP is less common in recent years,and it is often mixed with other pathogens and cause porcine respiratory disease syndrome(PRDC).Because of the unsatisfactory cross-protection effect between different serotypes of APP,it has increased the difficulty of the prevention and treatment.In addition,the disease is prevalent in many pig-raising countries and has become one of the five major epidemics of industrialized pig-raising in the world,seriously affecting the development of the pig industry and causing huge economic losses.In order to enrich APP etiology and genomics data and develop an APP vaccine with good cross-immunity protection.In this study,a suspected Actinobacillus pleuropneumoniae strain was isolated from the lungs of a dead pig with respiratory disease.Observation of culture characteristics,biochemical identification and molecular biological identification determined that the isolate was Actinobacillus pleuropneumoniae,named SDHG-1.Growth curve measurement,surface polysaccharide PGA transcription factor detection and determination of PAM adhesion and invasion ability were illustrated the biological characteristics of SDHG-1.And whole-genome sequencing and comparative genomics method were used to reveal the characteristics of the genome sequence of SDHG-1.Finally,this study also detect the pathogenicity of SDHG-1 to mice and piglets and immunogenicity.The results showed that SDHG-1 is an APP isolate with special culture characteristics,forming"adhesive"colonies in solid plates.It is highly sensitive to streptomycin,fosfomycin,ceftriaxone sodium,florfenicol,enrofloxacin,and ceftazidime.The results of biological characteristics show that the concentration(Y)of the bacterial solution between 4h-8h of culture(OD600?0.3-1.5)is extremely significantly correlated with the OD600 of the bacterial solution.Their regression equation is Y(CFU/m L)=5×10¹¹×OD^6.1396.The transcription levels of the isolated extracellular matrix polysaccharide PGA synthesis genes pga A and pga C were significantly higher than that of the non-sticky strain L20;the adhesion and invasion results showed that the adhesion and invasion ability of SDHG-1 to PAM is stronger than that of APP L20.Genome sequencing results show that the SDHG-1 genome has a total of 23,22048 bp,with a GC content of 41.22%,and a total of 2172 genes are encoded.In the middle of the genome,a tetracycline-resistant gene island was obtained by horizontal transfer,and the difference analysis of virulence genes with APP L20found that SDHG-1 lesion induction related virulence factors accounted for the largest proportion,while the L20 strain accounted for the largest proportion of nutrition acquisition Related virulence factors,which may provide new ideas for the virulence difference between traditional serotype virulent strains and newly emerging strains.In addition,SDHG-1 is pathogenic to mice and piglets:SDHG-1 infected mice can cause breathing difficulties,messy coat,anorexia,and depression.After 6 hours of death,the lungs were severely congested and hemorrhage,according to Kou's rule,the LD₅₀ of SDHG-1 to mice is 7.1×10⁹CFU;after 48 hours of infection,the piglets have runny nose and panting.Autopsy reveals fleshy lung lesions with typical fibrin-like exudates on the surface.Inactivated SDHG-1 can produce 40%protection against serotype 1APP infection in mice,but it cannot provide effective protection against serotype 5b APP infection.In summary,this study successfully isolated a viscous serotype 12 APP SDHG-1with abundant expression of PGA on the surface;SDHG-1 has inserted a tetracycline-resistant gene island obtained by horizontal transfer;this strain had strong pathogenicity to piglets.Inactivated SDHG-1 provides a better immune effect against self-infection and a good cross-immune protection effect against serotype 1 APP infection.It is a potential vaccine candidate strain.