| Objective:Study Campylobacter jejuni(Campylobacter jejuni,Cj)different virulence among isolates colonizing Gram stain morphology and growth and reproduction ability,athletic ability,as well as understand the C.jejuni isolates virulence variation after continuous multi group mice were gavaged with digestive tract.Obtaining separated Cj after Campylobacter jejuni colonization and discharge in the digestive tract.We compared with the original generation of Campylobacter jejuni.And Understanding and awareness of the ability of the different virulence of Campylobacter jejuni isolates induced peripheral neuropathy.Methods:1 Recovery and culture primary Campylobacter jejuni:The Campylobacter jejuni strains which was Selectd from the Department of Neurology Lab-80 ℃ refrigerator frozen the Second Hospital of Hebei Medical University,and isolated from the stool of acute motor axonal(AMAN type)Guillain-Barre syndrome(Guillain-Barre syndrome,GBS)patients called Campylobacter jejuni strains lulei.Remove the Cj from-80 ℃ to-20 ℃ refrigerator freezer,and then from-20 ℃ to-4 ℃ refrigerator freezer,after a few minutes at room temperature,the Cj melted.The bacteria liquid was poured into the flask containing 5ml sterilized broth culture medium,which was placed in the three gas incubator and enrichmented 24 hours at 42 ℃ microaerophilic(10% CO2,5% O2,85% N2)Environmental.The next generation was passaged to the 7% defibrinated sheep blood agar of Columbia,and cultured in the same environment for 24 hours.In the same way to the third generation,the successful recovery of Campylobacter jejuni.2 Identification of Campylobacter jejuni: Using Gram stain、Sodium hippurate hydrolysis test and Multiple PCR method to Identify the Campylobacter jejuni.3 Selecting and grouping method of experimental animals: Select 4 weeks old,weighing 10-12 g,clean healthy male mice.They were divided into 18 batches,each batch of 6 mice.4 Isolation of C.jejuni from the mice feces before filling the stomach.5 Gavage manner and concentration: The viable bacterial suspension was 3 × 108 cfu / ml concentration of 0.5ml through oral gavage.All mice were fasted before gavage for 12 hours and drunk water immediately after the administration.6 Isolated and cultured of Campylobacter jejuni: first batch mice was gavaged the original strains of Campylobacter jejuni lulei with 1 time.Mice feces were collected at 24 hours and 7 days,8 days and 9 days after irrigation.And isolated and purified Campylobacter jejuni isolated mutants from the feces of the above time points colonization.The separation and purification of C.jejuni lulei of isolated mutant with 24 hours was fed the second batch of mice.And so on,until the completion of the isolation and culture C.jejuni of the 18 batches.7 The growth and reproduction ability,movement ability and the change of the morphology of the gram stain of the isolated strains of C.jejuni in different time points of each batch: The growth and reproduction ability,movement ability and morphological changes of bacteria are important indexes to evaluate the virulence of bacteria,The differences of growth and reproduction ability,movement ability and the change of the morphology of the different strains were compared by using bacteria nephelometry,sports ring diameter Gram staining method.8 Using statistical software SPSS 13.0 to analyze the growth rate of the isolated strains of C.jejuni and the diameter of the moving ring in each time point of each batch.9 Observation of the general situation of experimental animals: the daily status of the mice were observed since the date of purchase : Such as eating water conditions,mental status,stool,fur smoothness,body weight and physical activity,with or without diarrhea and limb paralysis and other symptoms.The average observation time was 30 days,and then the animals were sacrificed anatomy.The animal appeared obvious symptoms of paralysis within 30 days,at any time the bilateral sciatic nerve were dissected and pathology observed.Results: 1 The successful recovery of primary Campylobacter jejuni lulei;2 Campylobacter jejuni was not selectd from the faeces of the Kunming mice before filling the stomach;3 Obtaining Campylobacter jejuni isolation first batches to eighteenth 1batches of 24 hours;4 The isolation of colonization of Campylobacter jejuni were not abtain within seven days,eight days,nine days of the first batch to the seventh batch;5 The Campylobacter jejuni can colonize in the mice digestive tract since the beginning of the eighth batch;6 Obtaining the isolation of colonization of Campylobacter jejuni within seven days,eight days,nine days of the eighth batch to the eighteen batch;7 Statistical software SPSS13.0 analysis for each time point of each batch,using Kruskal Wall Test,LSD analysis different batches of the same time point of the isolation of colonization of C.jejuni.There are differences in growth and reproduction rate per unit time between different batches of the same time point of the isolation of colonization of C.jejuni,the growth and reproduction capacity of the isolation of C.jejuni is gradually increased which compared with primary Campylobacter jejuni with the increase in batch,there are differences about growth and reproduction capability of the isolation of C.jejuni between different batches;we analysised the different time points of the same batch of the isolation of C.jejuni with repeated measures design analysis,there are differences in growth and reproduction rate per unit of time at different time points between the isolation of the same batch;8 Statistical software SPSS13.0 analysis for each time point of each batch,using Kruskal Wall Test,LSD analysis different batches of the same time point of the isolation of colonization of C.jejuni.There are differences in movement ability between different batches of the same time point of the isolation of colonization of C.jejuni,the movement ability of the isolation of C.jejuni fluctuated within a certain range which compared with primary Campylobacter jejuni with the increase in batch,there are differences about movement ability of the isolation of C.jejuni between different batches;we analysised the different time points of the same batch of the isolation of C.jejuni with repeated measures design analysis,there are differences in movement ability at different time points between the isolation of the same batch;9 We observed meorphology changes of different batches of the same time point of the isolation of colonization of C.jejuni through Gram staining.With the increase of the batch,the body of the isolation of C.jejuni gradually became longer and thinner,the degree of bending rod-shaped or rod-shaped spiral became clear,with double "S" shaped or three "S" shape;we analysised the different time points of the same batch of the isolation of C.jejuni through Gram staining,the body changed.With increasing time points,the body of the isolation of C.jejuni gradually became longer and thinner,the degree of bending rod-shaped or rod-shaped spiral became clear;10 Observation of the general situation of experimental animals: When the mice were sacrificed 30 days,the ability of activity,drinking water and eating of the mice as usual,having well mental state,body weight had not reduced significantly,diarrhea and paralysis and other symptoms did not appear.Conclusions:1 Campylobacter jejuni was not detected in the stool of the Kunming male clean grade mice in the Animal Center of Hebei Medical University.2 Obtaining the isolation of C.jejuni which was fed the mice in the first 24 hours.3 Campylobacter jejuni can colonize in vivo in mice with feding one time.4 Each batch could not be isolated,after 7,8 and 9 days of the mice were fed with C.jejuni one time.5 The morphology of the isolated strains of C.jejuni changed by the culture of the mice,which were shorter and shorter,the curved or spiral rods were more obvious.6 The growth and reproduction rate of the isolated strain of C.jejuni in vivo culture was faster.7 Most of the movement ability of the isolated strain of C.jejuni enhanced in vivo.8 C.jejuni were cultured in mice with feding one time.The mice had not diarrhea,paralysis and other clinical symptoms in 30 days. |
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