| Trichinosis is a foodborne parasitic disease caused by Trichinella spiralis that usually parasitizes in intestinal tract and striated muscles of host.Pork and its products are the main sources of infecting Trichinosis.Trichinella can infect human and more than 150 other animals.According to statistics,almost 15 million people worldwide are infected with Trichinella.China is one of a few countries in the world that have been most seriously affected by this disease.In 2009,the Ministry of Agriculture of China classified Trichinosis into the “26 most dangerous zoonosis” list.Intestinal helmith and intestinal microflora coexist in intestine;helmith infection can affect the intestinal microbiota distribution ofhost,while changes of intestinal microflora also have an impact on the quantity and adaptability of helmith.In this study,the effects of intestinal microflora changes on the infection of Trichinella spiralis and on immune response in mice were taken as the targets.The intestine dysbacteriosis model was first established using ceftriaxone sodium antibiotics;PCR-DGGE and viable counts were used to analyze the rotation.Trichinellosis infections were analyzed using parasite counts and histopathological methods,and flow cytometry was used to detect changes in immune parameters in mice to study the intestinal bacteria in mice under antibiotic stress.The results are as follows:(1)Establishment of intestinal microflora dysbacteriosis mice model under the pressure of antibiotics: 45 BALB/c mice were randomly divided into three groups: TS group,blank group,and antibiotic intervention group;mice in antibiotic intervention group was administered with ceftriaxone sodium(5g/kg)twice for 8 days and feces were collected;changes in intestinal flora were observed by PCR-DGGE and colony counts.The results of PCR-DGGE showed that the difference in similarity coefficient was obvious and richness decreased markedly among antibiotic intervention group,TS group and blank group,and there was a significant difference in phylogenetic tree among antibiotics intervention group,blank group and TS group.Colony counting showed that the total number of Enterobacteriaceae,Enterococci,Bifidobacteria,and Lactobacillus significantly declined;Bifidobacterium,Enterobacteriaceae,and Lactobacillus fell 3-4 orders of magnitude in feces,which were significantly lower than that of normal mice.The above results indicated that the intestinal microflora dysbacteriosis mice model has been successfully established.(2)Effects of intestinal microflora changes on Trichinella spiralis parasitizing and immune response in mice: 45 female BALB/c mice were randomly divided into three groups with 15 mice in each: namely Trichinella spiralis(TS)infection group,blank group,and antibiotics intervention group.The mice in the antibiotic intervention group were twice given ceftriaxone sodium(5 g/kg)for 8 days,and after that,each mouse in TS group and antibiotic intervention group was fed with 300 Trichinella spiralis muscle larvae.Three groups of mice feces were collected on 7th,14 th,21st,28 th,and 35 th days after infection.Changes in intestinal flora were observed by PCR-DGGE and colony counts;7-day intestinal tract adult mice expression changes of Th1(IFN-γ)and Th2(IL-4,IL-10)cytokines in infectious mice were detected by flow cytometry;the intestinal and tongue muscles were used to make pathological sections and observe the intestinal and lingual muscle pathological changes.Finally,based on the results of bacterial colony counting,four common intestinal bacteria were mixed with adult Trichinella spiralis and cultured together to observe adult motility and incubation rate.The test results showed that microflora in antibiotic intervention group gradually increased after infected with Trichinella spiralis,at 35 days,and the number of microbiota was approaching to that of blank group.Colony counting showed that the number of Lactobacillus and Bifidobacterium in TS group was lower than that of blank group on days 7,14,21,and 28 with significant differences(P<0.05),but there was no significant difference on the 35 th day(P>0.05).After comparing the microbiota containing Lactobacillus,Bifidobacterium,Enterococcus,Enterobacteriaceae in mice ofantibiotic intervention group,and blank group,Lactobacillus and Bifidobacteria were lower than that of blank group at 7,14,21,28,and 35 days with significant difference(P<0.05),Enterobacteriaceae and Enterococcushad no significant differences at 35 days(P> 0.05).The number of adult and muscle larvae in antibiotic intervention group was lower than that of TS group with significant difference(P<0.05);LPG and RCI were also lower than that of TS group with significant difference(P<0.05).Flow cytometry detection of cytokines showed that the secretion of Th2 cytokines IL-4 and IL-10 in the TS group was higher than that of blank group with significant difference(P<0.05),but there was no difference at 35 th days compared with blank group(P>0.05);Th2 cytokines IL-4,IL-10 in antibiotic intervention group were higher than blank group,but the difference was not significant(P> 0.05);the secretion of Th1 cytokine IFN-γ in TS group was lower than blank group,and the difference was significant(P<0.05).There was no difference on 35 days after infection compared with TS group(P>0.05).The level of IFN-γ secretion in antibiotic intervention group was lower than that in blank group,but there was no difference(P>0.05).The results of duodenal pathological sections showed that inflammation in TS group was more serious than that of antibiotic intervention group on 7,14,21 days,and pathological changes were similar on 28 and 35 days,which were close to equilibrium.The number of Trichinella cystsin tongue in TS group was more than that of antibiotic intervention group.The results of adult and bacterial medium showed that Lactobacillus,Bifidobacterium and Enterococcus spp.play a promoting role in the process adult inoculating newborn larvae compared with the negative control,and the difference was significant(P<0.05).E.coli had an inhibitory effect on adult inoculating larvae with a significant difference(P<0.05).Through the above research,we found that microflora changes can affect the establishment of Trichinella spiralis parasitic life and immune response level.In particular,Lactobacilli,Bifidobacteria,and Enterococci in intestine are conducive to the parasitism of Trichinella spiralis.Enterobacteriaceae hampers Trichinella spiralis parasitizing in the intestine.The research will provide a theoretical reference for the relationship between intestinal microflora and parasitic infection. |
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