Establishment Of Animal Infection Model Of Actinobacillus Suis And Study On Its Virulence Factors

Objective:Actinobacillus suis(A.suis)is an important conditional pathogen that can colonize on the upper respiratory tract of healthy pigs of different ages.And A.suis can spread vertically from sows causeing sudden death of suckling piglets.In recent years,A.suis has gradually become a potential risk for highly healthy pigs of all ages.It has even been reported that people bitten by diseased pig was identified to be infected with A.suis.At present,there were few studies on A.suis in China,especially the research on the pathogenic mechanism and virulence factors of A.suis.Therefore,in this study,two strains of A.suis standard strains were selected for further research.By constructing A.suis infection models for mice,yellow rabbits and pigs,and performing pathological histological studies on these model animals,the pathological changes and pathogenesis of A.suis were explored.In addition,some virulence factors of A.suis were also selected to study their differences in the expression of three model animals in vitro and in vivo to explore the role of different virulence factors of A.suis in the pathogenesis.Method:A.suis ATCC15557 and ATCC33415 were infected with mice,yellow rabbits and pigs respectively by intraperitoneal injection to construct the animal infection model.During this period,the clinical symptoms of model animals were constantly observed,blood was drawn and body temperature was measured at regular intervals,the dynamic changes of blood immune cells were monitored as well.Finally,the morbid animal model was examined by necropsy.A.suis was isolated and identified from various organs of the model animal and pathological tissue sections were made to study the pathological damages.Real-time fluorescence quantitative PCR was used to study the relative expression levels of virulence related genes such as ashA,ompA,apx?,and hugZ genes in major visceral organs(liver,spleen,lung,kidney)of A.suis in vitro and in model animals.Result:1.Both A.suis strains can infect mice,yellow rabbits and pigs and cause death.The LD50 of A.suis ATCC15557 to mice is 1.265 × 108 CFU,and A.suis ATCC33415 to mice is 2.350 ×108 CFU.2.Model animals had similar clinical symptoms after infection,with the most obvious symptoms in the infected pigs.The main clinical manifestations of model animals were rapid onset,cough and tremor,inconvenience in mobility,and with high fever.The necropsy showed that most of the model animals had ascites,irregular small petechia in the kidneys,and a large amount,of fibrin in the lungs.The tissue sections showed necrosis and inflammatory cell infiltration in various diseased organs;the lesions were most pronounced in animal lungs.A.suis strains can be isolated from the main organs(liver,spleen,lung,and kidney)of model animals that died of the disease.3.In general,the relative expression levels of the virulence factor in the two A.suis strains were generally higher than other virulence factors both in vitro and in the organs of the model animals.Under the same in vivo or in vitro culture conditions,the expression level of most virulence factors of A.suis ATCC15557 was slightly higher than that of A.suis ATCC33415.From the expression of A.suis single virulence genes in various organs of model animals,the apx? gene is abundantly expressed in the lungs of model animals;the ashC gene had higher expression levels in the liver,spleen,lung and kidney of the three model animals.The relative expression level of ashC gene was higher than that of ashA gene in the organs of the model animals and it was only lower than that of ompA gene,which was contrary to the in vitro expression,indicating that the ashC gene may play a more important role than the ashA gene in animal pathogenic processes.The hugZ gene and hgbA gene were mainly expressed in the livers and spleens of model animals.Conclusion:1.The pathogenicity of A.suis ATCC15557 was slightly higher than that of A.suis ATCC33415.They all have strong pathogenicity to mice,yellow rabbits and pigs,and infected animals usually have a faster onset.2.A.suis can cause various degrees of damages to the liver,spleen,lungs,and kidneys of animals,with the most serious damage to the lungs.3.Some virulence genes of A.suis have different expressions in vitro and in vivo.The relative expression of ashA gene in vitro was higher than that of ashC gene,but the relative expression of ashA gene in animals was lower than that in ashC.The relative expression levels of apx? gene,hugZ gene and hgbA gene were lower in vitro,but apx? gene was abundantly expressed in the lungs of model animals;hugZ gene and hgbA gene were abundantly expressed in the liver of model animals.Which explaining that some virulence genes of A.suis need to be controlled under certain regulation.4.The relative expression levels of ompA gene in vitro and in model animals were significantly higher than other virulence factors,which indicating that it may play an important role in the process of A.suis virulence.