| Pseudorabies(PR)is one of the most significant infectious diseases in the pig industry,and its causative agent is Suid Herpesvirus Type 1(SuHV-1).The symptoms include various degrees of respiratory distress,nervous and genital disorders,and mortality,according to the age of the host and the virulence of the virus strain involved.In recent years,the emergence of mutated strain makes it more difficult to prevent PR.Bacillus subtilis is a probiotics,which can depress some kinds bacterium and epiphte.There are few report on the antiviral effect of Bacillus subtilis.Therefore,this study was carried out in order to provide a basis for the application of Bacillus subtilis in animal husbandry.A strain of Bacillus subtilis,which we named CCBS-1,was identified by 16 SrDNA and gas chromatograph,which can depress Staphylococcus aureus and Escherichia coli.Extracted crude protein from the fermentation broth by acid precipitation separation and methanol extraction.At the same time tested the the concentration of extractive.The fermentation broth and two kinds of crude extra PRV were used to the invitro inhibition test of Suid Herpesuirus Type 1.After a serious of safe concentration,blocking,inhibition and killing test,we found that the crude extra PRV of fermentation broth and protein cam depress SHV-1 under the concentration of0.042mg/ml and 0.167mg/ml,and they can kill SHV-1 under the concentration of 0.111mg/ml.In this study,we used in vivo antiviral test of Bacillus subtilis in Kunming mice.24 rats from 18 g to 22 g were randomly divided into four groups: PBS control group(group PBS),Bacillus subtilis prevention group(group BS),and SHV-1 positive group(group PRV).Firstly,After 7 days,group BS were fed with 1.2×108CFU of Bacillus subtilis for each mice in a period of 14 days.Group PBS and group PRV were given the same dose of PBS.Secondly,we collected blood from caudal vein of every mice in Day 7.After 14 days we randomly choose 4mice of PBS group and BS group for immune organ index text by using their spleens and thymus.Thirdly,spleen cells were taken for lymphocyte transforming test,quantitative detection of CD3+CD4+ and CD3+CD8+ T cell subpopulation,non-specific antibody detetion of IgG,IL-4,IFN-? seoretion level detection and the detection of NK cell activity.At the same time,we used the serum,collected in Day7 and Day 14,for antibody IL-4 and IFN-? level determination.Finally,group PRV and group BS were infection with SHV-1(106.70 TCID50).After the death of Kunming rats,brain tissues were taken to make pathological sections.The results indicated:(1)The weight of Kunming mice in the groups that fed with Bacillus subtilis were significantly higher.(2)The thymus index of group BS was significantly higher than groupPBS,but the spleen index was no significant difference.The thymus index and spleen index of group BS were no significant difference with group PBS.(3)The specific non-antibody levels of three groups showed an upward trend.The level of Day 14 was higher than Day 7 and significantly higher than group PBS.(4)The stimulation index of group BS was higher than that of group PBS.(5)The group BS promoted the proliferation of CD3+CD4+ and CD3+CD8+ T lymphocytes.(6)The activity of NK cells group BS was significantly higher than that in the group PBS,the activity of NK cells was increased by 25%.(7)The levels of IL-4 and IFN-? ray in serum and supernatant of group BS were significantly higher than those in the PBS group.(8)The rate of BS prevention group attack is 25%.(9)There were obvious edema,hemorrhage and inflammatory infiltration in the attack group,but there was no such phenomenon in the group BS.Based on the above results,The fermentation broth of Bacillus subtilis CCBS-1 strain had a certain inhibitory effect on SHV-1.These strain can improve the humoral immunity and cellular immunity in animal and have preventive effect on Swine pseudorabies. |
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