| In order to do research on excreting regulation of Isospora suis oocysts infected with piglets by oral infecting,we analysed data of nine artifical infections of purified I.Suis.The results showed that:(1)The prepatent periods are always 5 days, and the patent periods are significantly different,rangeing form 4 days to 7 days.(2) The second oocysts excreting peak appeared 1 or 2 days after the first occysts excreting peak in some experimental groups.(3)Thin wall and thick wall type oocysts first appeared after oocysts sporulated.(4)There are thinner wall type oocysts at the end of the patent periods.In order to reveal the dynamic changes of the T lymphocyte subgroups in piglets artificaially infected with I.suis,nine six-day-old piglets were innoculated with I.suis sporulated occysts,and CD3~+,CD4~+,CD8~+ T cells were checked on before and post inoculation 1,3,6,9,11(DPI),respectively.Results showed that the amounts of CD3~+,CD4~+ T cells increased on DPI 3 and then decreased,but the difference was not significant(p＜0.1);however,CD8~+ T cell raised on DPI 6 and reached the peak value on DPI 9(P＜0.05).The ratio of CD4~+/CD8~+ began to descend on DPI 6,and came to the lowest point on DPI 11,which indicated that the immunological functions of the piglets infected with I.Suis were suppressed.Two sets of nested PCR primers were designed by bio-software to amplify 18S rRNA gene of Isospora sp.after searching in GenBank for the corelated sequences of Isospora sp.18S rRNA gene.The data of the experiments showed that both of the two sets of nested primers could amplify the target fragments from lots of purified oocysts,and they also could get the target fragments from small volume DNA templates which were diluted to be equal to less than single oocyst DNA content. The results showed that the primers were very sensitive.The two sets of nested primers were used to amplify the DNA samples extracted directly from stool samples colleted from a farm.The results showed that the second set of nested primers was better than the first set of nested primers in specificity,and it's also suggested that the nested PCR method were more sensitive than the MacMaster's method.Artifical positive stool samples which were made by mixing the negative stools with quantita te oocysts were used to test the two sets of nested primers.The data of the experiment suggested that the second set of nested primers could amplify DNA samples which were extracted directly from stool samples contenting small volum of oocysts.Five different DNA extracting methods,method CTAB,method BC,method TT, method MK and method MMK,were designed to screen a better method to extract DNA from small volume oocysts.The DNA samples extracted were detected by the second set of nested primers and spectrophotometer.Method CTAB was concluded as the best method for small volume oocysts DNA extracting,and method TT was better than the method MK and MMK.Method BC was failed to extract DNA for nested PCR amplification.The results from the spectrophotometer confirmed the results from the PCR reaction.It showed that the method CTAB was suitable for small volume oocysts DNA extracting and the downstream PCR reaction.In this experiment a DNA extracting method for small volume oocysts were screened and a set of nested PCR primers sensitive and specific enough to amplify the DNA extracted directly from stool samples were designed.The DNA extracting method and nested PCR primers would be used in future research.
|
PDF Full Text Request