Research On The Change About Virulence And Ability To Cause Peripheral Neuropathy After Campylobacter Jejuni Through The Digestive Tract In Mice

Objective: Compared with the original generation of campylobacterjejuni, to study the different sources of campylobacter jejuni after a series ofthrough the digestive tract of the mice，the difference between the variants ofthe growth and reproduction ability, the change about Gram staining andmorphological. At the same time, to further observation and analysis theability of campylobacter jejuni cause the difference of peripheral neuropathycaused by variation strains,to lay a foundation for understanding the changesin virulence about C.jejuni in gastrointestinal environment and the differencesin peripheral neuropathy induced by C.jejuni with different virulence.Method:1.Recovery and culture C.jejuni：the C.jejuni lulei strain isolated fromacute motor axonal of Guillain Barre syndrome（GBS）and C.jejuni NCTC11168strain that isolated from the faece of bacterial diarrhea patient，the twostrains were storaged in-80℃refrigerator in the department of neurology ofthe second hospital of Hebei Medical University. They were melted at roomtemperatyre, then took500ul bacteria liquid into a sterilized5ml clean bottlewith Brucella broth, took the bottle into a three gases incubator, in42℃microaerophilic environment enrichment for24hours, then generation toColumbia blood plate containing7%defiber sheep blood, at the sameenvironment for24hours, with the same way for the third generation tosuccessfully recover C.jejuni.2.Identification of C.jejuni：by using the method of Gram stain and sodiumhippurate hydrolysis test to identify C.jejuni.3.To choose experimental animals and divide into groups：choose4weeksof age, weight18-20g, healthy and male Kunming mice with clean grade. Divided the mice into9batches, each batch with3groups：lulei group、NCTC11168group and Brucella broth control group，2mice in each group, a tatalof54mice.4.Method and concentrain：the experimental group mice were orally activebacteria0.5ml with3×108cfu/ml，the control group mice were orally0.5mlsterilized Brucella broth. All mice fasted12houred before intragastric, thengive fresh water and food immediately.5.Isolate and culture C.jejuni: the primary C.jejuni lulei and NCTC11168strain fed the first batch, intragastric twice, apart24hours, collection micefaece in48hours and72hours after intragastric administration, then isolatedand purification C.jejuni from faece. The purified72hours C.jejuni wereorally next batch., until to the ninth batch.6.To observe the change of the C.jejuni in growth ability and Gramstaining in different batches：the ability of growth and reproduction of bacteriais an important index to evaluate the virulence of bacteria. Respectively usingthe method of plate counting and Gram staining to compare the ability ofgrowth and reproduction and the change of morphology between the C.jejuniisolated strains.7.To observe the general condition of experimental animals：mice weremonitored daily from the date of intragastric, such as the situation of intakefood and water, mental state, stool character, body weight, physical activityand so on. General observation period was30days, then autopsy the mice, thebilateral sciatic nerves were observed by pathology, if the animal within30days with apparent paralysis, then autopsy the animal immediately.8.To examination the change of neuropathological in different batches ofmice by using the method of HE staining、immunohistochemical staining、osmic acid staining、 toluidine blue staining and transmission electronmicroscope.Result:1.Different batches of C.jejuni have different growth speed in unit time,each batch of the same point in time with the increaseing ability of growth and reproduction ability.2.Using the Gram staining, the different batches of C.jejuni havemorphological changes. With the increasing of the batches, the lulei strain ofC.jejuni become longer, thinner and spiral or curved rod-shape graduallyobviously, become a double “S”shape and three “S”shape; the NCTC11168strain of C.jejuni become gradually shorten, from a “straight rod”shape to a“C”shape.3.To observe the general condition of mice: the seventh batch of one rat inlulei group appear obviously pathological changes in sciatic nerve. That is tosay: HE staining showed uneven thickness and breaken in axonal.Immunohistochemical staining showed myelin disappear, axon uneventhickness and breaken. Osmic acid staining showed demyelination, myelinreaction,like a “wall” change, in near of the Ranvier section the changesobviously, at the same time, in near of the Ranvier section have visibleremyelination. Toluidine blue staining showed myslin loss and fracture, at thesame time, axon occurred like wallerian degeneration. Transmission electronmicroscope showed mitochondrial crista disappeared, membrance swelling,axonal membrance separation and disappearance of myelin membrane, myelinlamellar structure, a concentric like cable retraction to the axis direction. Theother mice had no obviously pathological changes.Conclusion:1.Different sources of C.jejuni lulei strain and NCTC11168strain bycontinuing intragastric administration, the ability of growth and reproductionwere enhanced, at the same time, bacterial morphology appearedcontinuously change. It means that the virulence and morphological ofC.jejuni increased after through digestive tract.2.After intragastric administration for2consecutive times, no obvioussymptoms of diarrhea disappeared, but it can appear similar to GBS paralysissymptoms and peripheral nerve pathological changes. Although GBS andC.jejuni has closely relationship, with the enhanced virulence of C.jejuni, theability to change the symptoms of paralysis and peripheral nerve pathology did not appear to strengthen. It means that to use the enhanced virulenceC.jejuni to gavage mice can not establish a stable animal model for peripheralneuropathy.