Study On The Differential Diagnosis And Pathogenicity Of Mycoplasma Hyorhinis And Mycoplasma Hyopneumoniae

Mycoplasma hyorhinis?Mhr?and Mycoplasma hyopneumoniae?Mhp?are important pathogens that caused Mycoplasma hyopneumoniae pneumonia.They are often synergistic with a variety of bacteria,viruses and environmental factors,causing porcine respiratory disease syndrome complex?PRDC?,leading to an increased case fatality rate,which has brought huge economic losses to the pig industry.The diagnosis of diseases caused by Mhr and Mhp requires preliminary diagnosis based on the epidemic characteristics,clinical symptoms and pathological changes of the disease,and laboratory tests are used to further confirm the diagnosis.At present,there is no effective differential diagnosis method of Mhr and Mhp,and there is no systematic comparative study on the difference of pathogenicity caused by the two pathogens in pigs.The aim of this study is to establish a laboratory diagnostic method that can distinguish these two pathogens,and make a comparative study on the differences in their pathogenicity,so as to provide technical means for the effective prevention and control of diseases caused by these two pathogens.Firstly,the whole Mhr was used as the immunogen,and a monoclonal antibody?mAb?1C4 was obtained by hybridoma technology.The ascites was purified by affinity chromatography and labeled with horseradish peroxidase?HRP?and an ELISA method capable of detecting Mhr antigen was established.The results of reproducibility test showed that the coefficient of intra-and inter-variations were less than 6%and the minimum detection quantity for Mhr antigen in this ELISA was 25 ng.Secondly,P37 is a membrane protein of Mhr that may be an important immunogen and is a potential target for diagnostic development.The recombinant P37 protein was successfully expressed using the baculovirus expression system,and the anti-Mhr mAb was prepared.IHC detection confirmed that the prepared mAbs could be used for the detection of Mhr in infected lung tissue.A panel of truncated P37 proteins was used to identify the minimal B cell linear epitopes of the protein based on these mAbs.The core epitope was determined to be ²⁰⁶KIKKAWNDKDWNTFRNF²²² and the epitope consists of two helices and one random curl.Thirdly,in order to establish a differential diagnosis method for Mhr and Mhp antigens,the whole Mhp and recombinant expressed Mhp-P46 protein as immunogens,and uses hybridoma technology to prepare specific monoclonal antibodies?mAb?against Mhp.The ascites was purified by affinity chromatography and labeled with HRP.An IHC staining method capable of detecting Mhp antigen was established.This method has good reactogenicity to Mhp antigen in lung tissue.There is no cross reaction to the detection of Mhr and PCV2 antigens,which can provide reliable technical means for the in vivo differential diagnosis of Mhr and Mhp antigens and the study of the pathogenic difference between them.Lastly,in order to investigate the difference in pathogenicity between Mhr and Mhp,Mhr-DL strain and Mhp-HLJ strain were used to inoculate pigs respectively.The clinical comparison,serum antibody detection,anatomical pathology and histopathological changes were evaluated.The results of pathological examination showed that all piglets in the Mhr inoculation group had multiple serositis and adhesion in the thorax.The lung tissues of piglets inoculated with Mhp showed different degrees of fleshy changes,and no cellulosic exudates were observed.The results of immunohistochemical examination showed that the distribution of Mhr and Mhp antigens was different in the lung tissues.The Mhr infected lung tissues showed a diffuse distribution,while the Mhp infection mainly concentrated on the bronchial mucosal surface.Under experimental conditions,both Mhr and Mhp inoculated pigs can cause different clinical manifestations and pathological changes,which provide a scientific basis for the study of the pathogenic mechanism of Mhr and Mhp.