Establishment Of PCR Method For Isolation And Identification Of Eperythrozoon Of Pigs And Experiment On Artificial Infection Of Eperythrozoon

Eperythrozoonosis （eperythrozoonosis） is eperythrozoon （mycoplasma spp.） parasitic on the surface of pig erythrocytes, plasma and bone marrow caused by fe-ver,anemiajaundice as the main clinical symptoms of zoonotic diseases. The disease can lead to acute or chronic infection in pigs suffering from infection, the main clinical manifestations of anemia, jaundice, fever and other symptoms, not only to reduce pig performance, and often mixed infection with other pathogens, causing huge economic losses to the pig industry.The study found that infected pigs Eperythrozoon divided into Mycoplasma suis and Mycoplasma parvum. To study in Shanghai swine Eperythrozoon epidemio-logy law, we design two pairs of specific primers based on references and verified with high sensitivity and good specificity, From Shanghai area two slaughterhouses, collecting a total of three times a swine anti-clotting, blood samples in total 736 copies, extracted the blood genomic DNA as template, Using two pairs of primers specific for a partial sequence of 16s RNA PCR amplification of gene、Cloning, sequencing and bioinformatics analysis. The resμLts show in a total of 736 parts of the sample, A total of 101 samples were detected Eperythrozoon positive,positive rate was 13.72%（101/756）,Where Mycoplasma parvum positive rate of 7.34%（54/7 36）, Mycoplasma suis positive rate of 4.76%（35/736）, The proportion of mixed in-fection was1.63%（12/736）,The positive difference of the sample two slaughterhouses third acquisition significantly （χ2=132.841, P<0.001）, In the present study,Jiadi-ng Area slaughterhouse positive rate of 2.41 percent in March, the positive rate of 33.21 percent in May, May was significantly higher than in March （χ2=81.484, P <0.001）In the Blood sample DNA amplification positive from the Shanghai area, Four Mycoplasma parvum specific primer amplification products were selected for seque-ncing and four Mycoplasma suis specific primer amplification products were subclo-ned into pMD18-T vector for sequencing, Sequencing resuLts showed that four Mycoplasma parvum show exactly the same sequence of amplified fragment,the len- gth is 483 bp, Sequence from BLASTn software online analysis obtained and in GenBank showed Mycoplasma parvum Indiana strains 16S rRNA gene （Accession No. NR₁21759）sequence homology to 100%,4 Mycoplasma suis show amplified fragment sequences are also exactly the same,the length is 482 bp. Sequence from BLASTn software online analysis obtained and in GenBank showed Mycoplas ma suis Morioka5、6、8 strains 16S rRNA gene （Accession No. AB610847、AB610 848、AB610849） and ZJ NB01 strains 16S rRNA gene （Accession No. KC907396.1） sequence homology to 100%, The homology with Mycoplasma parvum is 93.6%.Before the The experiment, we remove the spleen of Guangxi Bama miniature pig,then using the Positive blood infected with Mycoplasma parvum to inoculation the pigs, Blood samples were collected for specific PCR and sequence analysis, The results show that 2 of 5 detect to be positive after inoculation 12d,all to be positive after inoculation 15d, Artificially infected pig blood samples and inoculated with the original gene sequence DNA amplification product are the same. That sh-ows Model experimentally infected with Mycoplasma parvum successfully establishe d.The results of the study show that the existence of domestic pigs infected with Mycoplasma parvum, Mycoplasma parvum and Mycoplasma suis 16sRNA there are differences in the gene fragments. Through artificial inoculation, we firstly establish ed a model pig infected with Mycoplasma parvum,which is the foundation for the biological characteristics and pathogenicitof of Mycoplasma parvum.