Study Of Etiological Properties, Prevalent Situation And Its Host-pathogen Interaction Of Mycoplasma Suis

Haemotrophic Mycoplasma (hemoplasma) is a kind of uncultivated pathogen and obligates to parasite on the surface of erythrocytes of vertebrate hosts. The acute disease of hemoplasma in feeder pigs was described as febrile anemia and icterus with low morbidity and high mortality. Chronic hemoplasma infection results low reproductive efficiency in sows, growth retardation and increased incidence of complicating disease. This disease has induced big economic loss in pig farming. Due to its unsuccessful cultivation in cell free media, hemoplasma was not well recognized in medical field. Lots of research work, including genomic sequencing, identification of interesting protein, pathogenicity, drug and vaccine development and host-pathogen interaction, were not well studied.1. Etiology:Morphological confirmation of hemoplasmaThis study studied the morphology of hemoplasma by transmission and scaning electron microscope. The results showed the diameter of hemoplasma ranged from 0.2 to1μm. A 50nm wide spacer region that separated hemoplasma and red cells was identified by transmission microscope. Fibril-like organ was confirmed on hemoplasma. Some holes were left on red cell surface after hemoplasma organisms were disassociated. These holes would change the osmotic pressure of red cell significantly and cause final disruption of infected erythrocytes. Heat treatment in Tween-20 and EDTA buffer was efficiently for hemoplasma detachment. This study provided work foundation for downstream researches.2. Etiology: Shotgun proteomic analysis of hemoplasmaHemoplasma proteins were seperated by 1-D electrophoresis. Protein gel was divided into 4 portion and analyzed by MS/MS. Cross-identification confirmed 487 and 492 proteins were homology with Mycoplasma and Rickettsial agents. Resulting peptides were classified based on theirs molecular function. Metabolic pathway of hemoplasma was analyzed by mapping peptides in KEGG database. This study was expected to do contribution for its cultivation in vitro, biophysiology characterization, drug target confirmation and vaccine design.3. Host-pathogen interaction: An exploratory study confirmed protein of band 3 on erythrocyte as the binding candidate of hemoplasma invasionThis study is the first report on the host-pathogen interaction of hemoplasma. Hemoplasma membrane was purified by density gradient centrifugation. Membrane proteins of hemoplasma were labeled with either fluorescein or biotin. Binding of the fluorescein and biotin labeled proteins with enzyme-treated and untreated RBC was detected by fluorescence microscopy and western blotting. Results showed the binding was sialic acid-independent but trypsin and a-chymotrypsin sensitive. Erythrocytic membrane proteins that interact with hemoplasma were confirmed by co-immunoprecipitation assay. Protein information was obtained by LTQ-MS/MS. Results indicated proteins of band 3 and ankyrin were involved in the adhesion of M. suis on erythrocyte. Binding inhibition assay in vivo substantiated the blocking ability of band 3 against hemoplasma invasion.4. Epidemiology: The 1.8 Kb DNA fragment formerly confirmed as M. suis specific was originated from porcine genomeIn 2003, a study identified a 1.8 kb EcoRI DNA fragment as M. suis specific from a M. suis infected pig. However, in our laboratory, M. suis negative control samples from a health pig and PK 15 cell were all positive in PCR assay by which primers targeting on the 1.8Kb DNA fragment. We found that 1.8KB DNA fragment was highly similar with pig chromosome 14 (99% identity) via performing a sequence comparison in NCBI chromosome database. A 1.3 Kb flank sequence of that 1.8Kb fragment was obtained by genomic walking assay. The resulting 1376bp fragment also showed the homology with pig chromosome 14 (98% identity). Moreover, a pig specific repetitive sequence was identified in this flank sequence (91% identity). This study demonstrated the 1.8Kb DNA fragment was pig genome original but not M. suis specific.5. Epidemiology: Study of Hemoplasma Infection in Pig Farms in ShanghaiThis study aimed to investigate the infection situation of hemoplasma on pig farms in Shanghai, China. Blood samples of 172 pigs and 65 feeding workers or veterinaries were collected from 19 pig farms. Wright-Giemsa stained smear and electron microscopy were performed. Species specific PCR assay was developed and applied in the detection of M. suis. Results showed 86% of tested pigs and 49.2% of relevant workers were positive with hemoplasma infection. Close phylogenic relationship of the two isolates from swine and human was confirmed. Statistic analysis showed hemoplasma infection did not associate with gender, age and survey site.