Screening Of Probiotics From Swine Intestines And Their Antiviral Activities Against Tgev

The aim of this paper was screen of the anti-transmissible gastroenteritis (TGE) coronavirus probiotic strains from the porcine gastroint estinal tract and identification species. Then evaluated the anti-TGEV activity of the probiotics on swine testicular (ST) cells in vitro with3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)、TCID50、indirect immunofluorescence (IFT) and semiquantitative RT-PCR Designed to determine whether the probiotics isolated from pig intestinal had the role of against TGEV, evaluated the level of anti-viral, and settled the theoretical foundation for the future development of probiotics against diarrhea virus.In the experiments, a total of54bacterial strains isolated from the porcine gastrointestinal tract with MRS and named as R1-R54. At first, the antiviral effect of probiotic bacteria was determined as MTT in vitro and twelve probiotic bacterias had strong inhibition to TGEV. Eight probiotic bacterias were selected after pH2.0and0.3%bile concentration screen. Using biochemical identification and through16SrRNA gene sequences identify probiotic strains and the results dispiay that R3、R10、R15、R22、R38and R45were Lacbobacillus reuleri, R30was Lacbobacillus fermentumand and R51was Leuconostoc paramesenteroides.We determined the cell viability after treated by probiotics using MTT and analyzed the largest non-toxic concentration. we selected the highest sensitivity antibiotics on the eight probiotics by sensitivity test.According to the experimental results, we had the anti-virus research of probiotics in cells.The extracts of probiotic and metabolites were dilluted on the basis of the largest non-toxic concentration and diluted different concentration. The effect of anti-TGEV of probiotic and metabolites were studied in three ways which were adding probiotic/metabolites before adding virus, adding probiotic/metabolites and virus simultaneously and adding probiotic/metabolites after virus in ST and the live cell quantity was measured by MTT method. The inhibitory rate increased, the infection viral ability descend when the cells were pre-treated with probiotics; the cytopathic effect (CPE) of cells reduced when addition of probiotics and TGEV at the same time indicated that probiotics may have a direct role in destruction of the virus and have strain-specific. The lower inhibitory rate of viral replication when cells infected with TGEV before addition of probiotics to the cells suggested that the probiotics worked hardly after the cells were infected.Added probiotics and TGEV in test tube effected, analyzed the TCID50of TGEV in ST with Reed-Muench used the supernatant. The result indicated that probiotic has notable inhibitory action to TGEV. The TCID50/0.1mL was10-4.78,10-5.03,10-4.59,10-5.33,10-5.47,10-5.82,10-5.44,10-6.21respectively after prepaered by probiotic(10-6.46).Pre-treated ST with probiotics/metabolites and infected STwith TGEV. Detected the quantity of virus infected cell after2h with indirect immunofluorescence (IFT). The result indicated that probiotics/metabolites block the virus adsorbed the cells.Pre-treated ST with probiotics/metabolites90min and infected ST with TGEV. Detected the quantity of virus expression after24h with semiquantitative RT-PCR. The result indicated that probiotics/metabolites may be conglutination virus or play a direct role of damage the virus structure.In the experiments,8probiotics have anti-TGEV activity from porcine gastroint estinal trac and their probiotics/metabolites can not only destruct the virus, but also block the virus infection of the cells and inhibit the viral proliferation in the cells.