Inflammatory Cytokine Assay Of Pleuropneumonia Piglets And Actinobacillus Pleuropneumoniae Mutants' Pathogenicity Examination

Porcine pleuropneumonia(PP)is a common respiratory infection and one of the most important respiratory diseases in pigs,which has caused great economic losses to the pig industry.Porcine PP is caused by Actinobacillus pleuropneumoniae(APP).APP is a pig-specific,Gram-negative and capsular bacterial pathogen,which is mainly colonized in the upper respiratory tract.APP has a variety of virulence factors.When APP invades the body,the defense cells in the first defense line will produce a variety of pro-inflammatory cytokines.These pro-inflammatory cytokines are responsible for coordinating various humoral and cellular events to prevent the lungs from being infected by microorganisms.Previous studies have shown that APP infection can induce significant expression of cytokines IL-1,IL-6,IL-8 and TNF-? in porcine lung tissue.Although these cytokines are known to have multiple functions in regulating the immune response,excessive production of them can lead to lung damage in infected pigs.Porcine alveolar macrophage(PAM)is the most abundant innate immune cell in the distal end of porcine lung,which is located on the surface of porcine alveolar lumen.PAM first encounters pathogens that invade the body and helps coordinate the initiation of the immune response in the lungs and the elimination of pathogens.Pathogens invading the body can quickly activate PAM through the binding response of several pathogen recognition receptors.Activated PAM releases a variety of inflammatory cytokines and chemokines after a series of pro-inflammatory signaling,thus recruiting more immune cells to fight against pathogens,and this process involves a large number of complex inflammatory responses.Appropriate inflammatory response is good for the clearance of invasive pathogens,but excessive inflammatory reaction will cause damage to the body,and in severe cases,the damage is irreversible and even threatens to the life.Therefore,the purpose of this study is to find the related antigens that induce or inhibit the expression of inflammatory cytokines in APP pathogens,and make them become the key targets for new therapeutic drugs and new strategies of prevention and treatment.In order to achieve the above purpose,this study first consulted a large number of literatures and data,and used piglets as animal models for APP infection experiment to analyze the inflammatory factors of pleuropneumonia pigs.Then,according to the characteristics of porcine inflammatory factors in pleuropneumonia,by detecting the changes of mRNA levels of inflammatory cytokines in the same time after PAM was infected by APP mutant or non-mutant,the mutants with different induction ability were screened from the APP mariner transposon mutant library.Finally,according to the extent of induction ability change of the mutant strain,the corresponding mutant genes and related antigens,two interesting mutant strains were selected,and ICR mice were used to construct the infection model.The pathogenicity of the two mutants was examined by comparing and analyzing some indexes,including mortality,weight gain,clinical symptoms,lung pathological changes,lung index,pulmonary parenchyma bacterial loads and total protein content in bronchoalveolar lavage fluid(BALF).The results showed that after pigs were infected with APP,the clinical symptoms became more and more obvious and serious with the extension of time.At the same time,the expression of inflammatory cytokines such as IL-1,IL-4,IL-6,IL-8,TNF-?,IFN-? and GM-CSF were up-regulated in diseased pigs.Subsequently based on this phenomenon,12 mutants were selected from 200 APP mutants and their respective mutant genes and related antigens were identified.Finally,the mice experiments of APP mutant No.30,No.139 and non-mutant showed that in the early stage of infection,the pathogenicity of mutant No.139 to mice was quite high,and the mice were in a state of acute infection;In the middle stage of infection,the mutant No.30 still showed much lower pathogenicity than the mutant No.139 in most aspects,and even lower than APP 5b in some aspects.These findings provide a new perspective for the prevention and treatment of APP infection.