| Objectives: To study and compare the application of iodine staining and modified culture in the diagnosis of B.h.To observe the morphology of B.h and reveal the genotype of B.h positive specimen.To compare the growth of B.h between xenic cultivation and axenic cultivation.To establish SD rats infected model with axenic culture B.h and observe the pathological damage of colons in models.Observe the shift of Th1 / Th2 balance in rats with infection.Methods: The lowest detection limit,the growth curve and anaerobic environment formation time were compared between the common culture and the modified culture.Collected 397 stool samples from the Department of Outpatient Surgery,Affiliated Tumor Hospital,Guangxi Medical University.The samples were detected by iodine staining and modified culture.The positive specimens were stained with iodine staining and trichrome staining.The sensitivity,specificity,agreement rate and kappa value of iodine staining method were calculated by using the modified method as the gold standard.The genotyping was carried out by PCR.Use LES medium for xenic culture and combine with penicillin(the final concentration of 1000 U / ml),streptomycin(the final concentration of 1000 ug / ml),amphotericin B(the final concentration of 2.5 ug / ml),fosfomycin(the final concentration of 20 ug / ml),amoxicillin(the final concentration of 31.25 ug / ml)and chloramphenicol(the final concentration of 20 ug / ml)for axenic culture.For monoclone of B.h by using eggs supernatant-horse serum-Bacto agar mixed agar plate was tested.30 SD rats were divided into 5 groups,6 rats for each group.After immunosuppression by dexamethasone,the treatment groups were infected with 107 / mice.The levels of IFN-γ,TNF-α,IL-4 and IL in the serum were measured by enzyme-linked immunosorbent assay(ELISA).The rats in the control group wrer treated with the same volume of PBS.The rats were sacrificed at 0d,3d,6d,9d and 14 d.The expression of myeloperoxidase(MPO)in colon tissue was detected by colorimetric method.The pathological damage of colonic tissue and mesenteric lymph nodes was observed by HE staining.The expression of IFN-γ,TNF-α,IL-4 and IL-10 in colons were detected by fluorescence quantitative real time PCR.Results: The lowest detection limit of the modified culture was lower than the common culture,and the cells densities of modified culture were higher than the common culture in the first three days.In stool samples and short-term culture samples,vacuolar and granular forms was obeserved.Cysts was observed in stool samples.Significant difference of the positive rate between modified culture(5.54%)and iodine staining had statistical significance.The sensitivity of the iodine staining method was 18.18%,the specificity was 100%,the agreement rate was 95.47% and the kappa value was 29.57%.Vacuolar forms were the most predominant in the xenic culture and the axenic culture.The time for the peakdensity of the xenic culture and the axenic culture was almost the same.However,the density of xenic culture decreases rapidly,thedensity of axenic culture decreased slowly and maintained a high density.It was observed that egg supernatant-horse serum-Bacto agar mixed agar plate surface distributes “colony-like” white dots.Using invert the microscope to observe,and it was found that “colony-like” white dots were composed of a large number of vacuolar forms.The pathological scores of colon were all 0 point between the control group and the treatmented group.The content of MPO in infected rats was not statistically significant compared with that in the control group.The levels of IFN-γ and IL-4 in serum of infected rats were not statistically significant compared with those in control group.The level of TNF-α in the serum of the rats in the 14 d group was statistically significant compared with the control group(P=0.002).The levels of IL-10 in the serum of 3d group(P=0.003),6d group(P=0.013),9d group(P=0.004)and 14 d group(P=0.000)were statistically significant compared with the control group.The expression of IFN-γ in colonic group is statistically different(F = 144.979,P = 0.000).The 3d group,6d group and 14 d group are statistically significant(P<0.05)compared with control group.The levels of TNF-α in the colon are statistically significant(variance,welch test,F = 5.837,P = 0.021),but there was no significant difference between the two groups.The expression of IL-4 in colonic group is statistically different(F = 8.917,P = 0.006).The 6d group and 9d group were statistically significant(P≤0.05)compared with control group.The levels of IL-10 in the colon of each group were statistically significant(F = 3.575,P =0.023),and the 6d group was statistically significant(P<0.05)compared with the control group.Conclusion: The lowest detection limit of the modified culture was lower than the common culture,the sensitivity of the modified culture was higher than that of the iodine staining method,and it could be used for the laboratorydiagnosis of B.h.Combined with high doses of antibiotics could be used to axenic cultivation and solid culture medium could be used to monoclonal of B.h.The B.h of axenic culture was used to establish the B.h—infected animal model.ST3 type B.h infection could affect the immunoregulation of Th1 / Th2 in rats. |
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