Establishment Of PCR Method On Eperythrozoon Suis And Study On Eperythrozoonosis In Splenectomized Pigs

Eperythrozoonosis is a zoonosis (anthropozoonosis) in many kinds of animals, which attached to erythrocytes, dissociated in plasma and marrow. Formerly, E.suis is classified as the rickettsial agent. Phylogenetic analysis of 16S rRNA gene showed that eperythrozoon is closely related to mycoplasma recently.Eperythrozoonosis of swine mainly causes fever, icteroanaemia and embryonic death, is mixed with other diease that took difficulty for prevention and cure, makes largely economic losses. Blood smear examination is the mainly method of diagmosis, however, low number of organisms of early parasitemia or chronic infection can interfere with the effectiveness of this method, and the powder of dye similar with the parasite attached to the erythrocyte, which caused artificial positive. Inoculative splenectomize pigs is clearly diagmosed method, but it need much time. Serologic evidence of E.suis exposure include the indirect hemagglutinaton test (IHA), complement fixation test (CFT) and enzyme linked immunosorbent assay (ELISA), however, absence the regular of antibody change, artificial negative is constantly. In order to improve diagmostic procedure of E.suis, the PCR method was established.According to published genome sequence of eperythrozoon suis, a set of primers were designed, then a polymerase chain reaction was developed and 782bp fragment was amplified and sequenced. The similarity to the sequences published on Genbank was 99%. The specific test results showed that the 782 bp sequence was amplified only from E.suis, but not from normal porcine blood, genome of Toxoplasma gondii, Pseudorabies virus, Pasteurella multocida Actinobacillus Pleuropeumoniae , Haemophilus parasuis. It's indicated that the PCR assay is specificity. 106 blood samples of 15 pig farms in Hubei and Hunan province were examined using PCR assay, the highest positive rate reached to 83.3%, the mean positive rate was 26.4%.We also studied eperythrozoonosis in splenetomized pigs, a single infection with eperythrozoon suis in splenectomized pigs was described. Red skin, eyelid tumefaction, high temperature were observed in the splenectomize pigs with inoculated E.suis. Infected pigs didn't show anaemia with slightly change in RBC and Hb values, serum value of glucose declined, aspartate aminotransferase and alanine aminotransferase significantly increased. Semi-nested PCR diagnosed the blood of infected pigs was positive from the first day of post-infection. Parasitaemia wasn't obviously increased from Giemsa stained blood smear.The complete 16S rRNA gene of E.wenyonii-Hubei was determined by PCR. It was sequenced and compared with other sequences. The results showed that the sequence was98.7% identical to that of Neimark published. The 1471bp complete sequence was compared with 5 mycoplasmas, 14 haemotrophic bacterias and Anaplasma marginale available in GenBank. The data were analyzed and a phylogenetic tree was established. The results indicated that the E.wenyonii and other haemotrophic bacterias share a node with pneumoniae group > but the hereditary distance was far from Anaplasma marginale.