In Vitro Proliferation And Development, Application Of, Pcr Assay Of Mycoplasma Suis (eperythrozoon Suis )

In recent years, Swine eperythrozoonosis has prevailed widely in most areas of China, having aroused people's great attention. Once break, it would cause mass mortality of livestock and result in tremendous economic loss in breeding industry. in this paper the in vitro proliferation of Mycoplasma suis and the establishment of the PCR diagnosis method were carried out.Firstly, in vitro culture of Mycoplasma suis was carried out by using the medium prepared with the ratios of RPMI-1640 supplemented with 40% Fetal Bovine Serum (FBS) , 0.2% inosine and rabbit red blood cell (RBC) which acting as the in vitro culture carrier to multiplicate the Mycoplasma suis( M.suis).The in vitro maintenance was proceeded in the 37℃constant temperature and 5% CO2 condition. Through observing the earliest time of hemolytic and the highest infection rate, the optimal time for replacing medium and the optimal generation time were established. The result showed that the highest infection rates of rabbit RBC could be up to above 90% and the in vitro incubation could be constant passed exceeded 40 times by changing the medium every 24 hour (h) and passing one times every 36h. At 36h post-inoculation of the newly prepared porcine RBC naturally infected M.suis ,the peak infection ratio about 90% was reached and maintained until 96h post-inoculation.The germfree porcine RBC natually infected M.suis could be stored at 4℃for 30 day (d) without hemolysis. All the M.suis grew well before 96h in the incubation process, and only a few rabbit RBC hemolysis was happened after 96h. No obvious different rabbit RBC infection ratio was displayed by comparing the inoculating porcine RBC naturally infected M.suis with the newly prepared and stored at 4℃before 30d.Secondly, a specific PCR method was developed using the primers designed basing on the novel published genome DNA sequences of Mycoplasma suis (Eperythrozoon suis) to identify the suspectable M. suis infected pigs. And genome of pasteurella multocida, streptococcus suis, enteropathogenic e.coli, porcine toxoplasmosis, swine mycoplasma, salmonella, bovine Babesia Babesia, porcine Circovirus type II, blood of SPF pig were employed to identify the specificity of the PCR system. The PCR specificity was further evaluated by amplifying genome of 24 blood samples taken from suspicious infected pigs basing on the clinical signs and microscope check, and an expected PCR product about 666bp in length was amplified. The PCR product was subsequently cloned and sequenced, and the homology ratios of nucleotide and amino acids sequences of the PCR product with the corresponding region of published M.suis (AJ504999) genome were 98.19%,96.85%, respectively, which suggested that the Chinese M.suis strain have varied in genomic DNA comparing with the foreign strain. Variation position concentrated in the 43, 47~51, 57, suggesting that ORF2 may be M.suis functional structural gene.The methods of PCR diagnosis of Mycoplasma suis was successfully established in this study and it provide that the Chinese M.suis strain have varied in genomic DNA comparing with the foreign strain, which laid a foundation for the further research of M.suis genome,functional-structual protein and its antigencity. Also, the paper provided the scientific evidence for establishing the integrated prevention and control technology of M.suis.