Isolation And Identification Of Bordetella Bronchiseptica And Its Interaction With Class A Scavenger Receptors In Porcine Alveolar Macrophages

Bordetella bronchiseptica(Bb)is a leading cause of pig infection,for example,Bb is one of major pathogens which induce swine atrophic rhinitis.Furthermore,Bb is often seen in porcine respiratory diseases(PRDC).However,to date,How Bb can evade innate host defense for infection remains to be determined.Pig alveolar macrophages(PAMs),as the major immune cells,have the role of the first line in the lung.In fact,PAMs sense invading microbes and trigger innate immune response through innate receptors including TLRs,CLRs,and scavenger receptors.More and more studies showed that SRs,special class A scavenger receptors play a key role in recognization and phagocytosis of some bacteria.Yet,there is less known about pig class A scavenger receptor.Furthermore,to note whether or not class A scavenger receptor such as SRA and MARCO can mediate the recongnization and phagocytosis of Bb remain unknown.Objective: In order to further clarify the effect of Bb on PRDC and to gain insights in to macrophages-host interaction,we determine: 1)the status of Bb infection of pig lungs with PRDC;2)whether PAMs mediate recongnization and phagocytosis through SRA and MARCO.Methods:(1)first,collected respiratory symptoms typical of sick pigs from pig farms in Shanghai,killed pigs,stripped the lungs and sterile PBS lavaged,lavaged liquild centrifugal sedimentation collection,the precipitate coated the TSA serum plates,isolated bacterium;Blast analysis 16 s rRNA gene sequence was identified the Bb.PCR identification virulence factors of separation strains,virulence factors including pertussis toxin adhesion(PRN),dermonecrotizing toxin(DNT)and flagllin protein B(fla B).Finally,Bb was tested drugs sensitive experiment,total of 18 kinds of antibiotics,measured the bacteriostatic circle diameter and analysis of the results.(2)in the first place,Bb was recovery,purification and PCR identification,then Bb was labeled by FITC,fluorescence microscope to observe bacteria staining;Second,booked 8 healthy pigs of 30 day time,isolated lungs,lungs were lavaged by PBS and isolated PAMs,cells were blocked by drug Fucodian for class A scavenger receptor specificity sealants,differently processing cells to phagocytosis FITC labeled bacteria,the result was used by flow cytometry instrument analysis of different cells fluorescence signal intensity,statistical analysis results;Finally,recovery the cell that has been built stable expression MARCO,SRA and empty vector pCDNA3.0 cell lines,cells were to use G418 drug screening 4-5 generation,westernblot detected cell lines purpose gene expression,CHO-vector cell was served as negtive control,phagocytosis FITC labeled bacteria,enzyme standard instrument to detect fluorescent signal intensity,statistical analysis results.Results:(1)Isolation and identification of Bb and its characteristics: first of all,5 strains Bbs were isolated from 15 pigs which had been identified as PRDC.Second,we found all isolates had the selected virulence factors,important to Bb virulnece.Finally,drugs sensitive experiment was to use to test Bbs,all of isolates were found to be highly resistant to amoxicillin,ceftiofur,streptomycin,ampicillin and other 8 kinds of drugs.In contrast,all of them are sensitive to kanamycin,meropenem,gentamicin.In addition,some strains appeared resistant to erythromycin,florfenicol,doxycycline,or amikacin.(2)PAMs mediate recognization and phagocytosis of Bb through SRA and MARCO: First,we found that the universal blocker of class A scavenger receptor,named Fucodian,could effeciently inhibit Bb phagocytosis by PAMs by using of flow cytometry analsis.Second,we use the stable cell line expression SRA or MARCO to determine the role or SRA of MARCO in phagocytosis of Bb.Conclusion: Our finding demonstrated that Bb infections play important role in PRDC.Furthermore,our data showed that PAMs mediate recognization and phagocytosis of Bb by SRA and MARCO.Finally,the alteration of functions of macrophages may facilate Bb lung infections and further prograss PRDC.Conclusively,our finding reveals that SRA and MARCO mediate recognization and phagocytosis of Bb,which is involved in PRDC.Furthermore,all of these resultes not only increase understanding PAMs-bacteria interactions,but also provide a basis of undstanding pathogenesis of PRDC.