Establishment Of Sandwich ELISA And Pathological Mice Model Against Mycoplasma Suis Infection

This paper established a double-antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) detection and mice infected model against Mycoplasma suis(M.suis) infection. Investigation of the transmit ways and pathogenicity of M.suis infection was performed in the present study by analysis of the clinic symptoms, pathological histology changes of mice model.1. Establishment of a double-antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for M.suis detectionThis study obtained pure M.suis by using water bath and filtration method. The M.suis antibody was prepared from experimental infected KM mouse and New Zealand rabbit with routine procedures. After optimizing reaction condition(mouse original antibody was diluted with 200 fold as the capture antibody,coating in 4℃overnight, blocking in 37℃for 1 h by glutin, and rabbit original antibody diluted 400 as the detection antibody, reacting in 37℃for 2 h, HRP labeled goat anti-rabbit antibody diluted 8000 fold, reacting in 37℃for 1 h, 37℃10 min for color appearance), and confirming by the sensitivity (minimum was 3.812μg/ml), specificity and repetitive experiments(coefficient of variation was 6.57 %,4.58 %), we established a steady sandwich ELISA for M.suis detection.The results of testing 91 swine's blood samples in Shanghai showed a 57.1 % positive rate by PCR, and a 34.1 % positive rate by blood smear microscopy while a 53.8 % positive rate by sandwich ELISA. It meant this method has the high feasibility. Since the crude antigen (blood lysis) can be used in this method, it provided a fast detection method against M.suis infection, especially suitable for the feeder works and veterinarians. 2. The preliminary research on the transmission way of M.suis infection In this report, the M.suis purified from positive swine's blood was used as source of infection. 20 SPF KM mice were used as experimental animal and randomly separated to several groups: intraperitioneal injection group, negative control group, water contaminated group, contacted group and blank control group.The infection in mice was confirmed by using microscopic observation, PCR analysis and sandwich ELISA detection methods. The results showed that only animals in the intraperitioneal injection group were infected with M.suis. It indicated M. suis might not be transmitted by ways of gastrointestinal tracts, air and breathing, saliva secretion easily. This conclusion would be helpful for further study.3. Establishment of mice model with pathological changes by M.suis infectionIn this research, the clinical symptoms, pathological changes and pathogeny were studied on the basis of mouse model infected with M.suis.The experimentally infected mice turn to be anorexia and breathlessness in our study. The results of pathological section showed obvious changes in the heart, liver, spleen, lung, kidney of infected mice but not in duodenum. The results of immunofluorescence assay showed all the fluorescence signals were distributed in the gap of tissue but not on the tissue cells, which elucidated the only target cell of M.suis attachment is erythrocyte.