Research On Peripheral Neuropathy Capacity Changes By Morphology And Virulence Of C.jejuni Via Gastrointestinal Successive Generations Pass Of KM Mice

Objective: After the successive generations pass of KM mice's digestive tract,the peripheral neuropathy capacity of Campylobacter jejuni(C.jejuni)has changed in morphology,growth rate and virulence.We expect to obtain enhanced virulence and stably genetic hereditary C.jejuni strains,which will increase the incidence of peripheral neuropathy in animal model and further improve this model to a better one.Simultaneously,it can offer experimental data on pathogenesis of C.jejuni to peripheral neuropathy.Methods:Recovery C.jejuni lulei strains and identify repeatedly.Take generation P4 as orally active bacteria to feed the first mice batch,and its concentrain is3×108cfu/ml.The experimental animals are cleaning Kunming(KM)mice about 4 weeks old.Collect mice feces at 24 hours and 48 hours after intragastric administration,then isolate and purify C.jejuni from feces as the first generation.The purified newly 24 hours C.jejuni are used to infect the next batch,until the eighteenth batch.Record mice's daily situation and compare morphology,growth rate and bacterial power measurement experiments of each generation to find the variation of them and the influence they have on peripheral neuropathy capacity changes.Results:1 We have succeeded in resuscitation of C.jejuni,which is identified to be negative by Gram stain and it seems like rod-shaped,commashaped or spiral-shaped under the light microscope.It is tested to be positive and dark purple by hydrolysis of sodium hippurate.And the identification by multiplex PCR gets two positive special bands of C.jejuni,one is 857 bp band for 16 S fragment and the other is 589 bp band for mapA fragment;2 Before intragastric infection,there is no C.jejuni being isolated from mice feces,whichindicates that experimental mice are not contagious with C.jejuni;3 The experiment has cultured eighteen generations successfully,among which the24 hours isolates are separated from the first generation and being stable from the eighth generation,the separation rate is more than 50%.Compared with 24 hours,48 hours isolates are separated from the seventh generation and being stable from the tenth generation,separation rate is more than 50%;4 Use Gram staining on each C.jejuni isolate and scrutinize the morphology under the light microscope.Most bacteria convert short-shape to elongated form and some individual C.jejuni morphology looks like type "C" or "S",along with spiraling more apparently;5 The growth rate of both 24 hours and 48 hours' isolates show a rising trend and achieve the maximum value in the eighteenth generation and the thirteenth generation respectively,and the maximum growth rate of 24 hours is higher than 48 hours,but in the whole view the growth rate of 48 hours is higher than that of 24 hours.Using SPSS 13.0 statistical software to analyze Kruskal Wallis test,LSD and one-sample t test,24 hours and 48 hours' growth rate are different and higher than that of primary generation.There are also differences between the two separation time points.24 hours and 48 hours' isolates have significantly changed from the eighteenth and seventh generation respectively.(P<0.05);6 C.jejuni isolates' power measurement experiment shows a increased tend approximately,24 hours and 48 hours isolates have achieved the maximum value at the eighteenth and ninth generation respectively.There are differences among each generations' bacteria loop diameter through Kruskal Wallis Test.The primary 24 hours' isolates differ significantly from the fifth generation.(P<0.05);7 During the experiment,the mice do not have diarrhea,paralysis,death,etc.Weaver's Score is 0;Bristol Stool Scale has got type four and five and there is no watery stool.The virulence of C.jejuni lulei strains has been enhanced through gastrointestinal passage and there is no clinical symptoms among the total 108 mice,which illustrates that KM male mice can be vivo medium of C.jejuni lulei strains and be of stability,repeatability and nonmortality.Conclusions:1 KM male mice can be vivo medium of C.jejuni lulei strains.2 The separation rate of C.jejuni becomes stabilized by successive generations pass via gastrointestinal tract.With the generation increasing,the isolates' growth rate and bacteria loop diameter show a growth trend.The virulence of 48 hours' isolates is stronger than that of 24 hours'.3 The short and small shapes of bacteria turn into elongated ones gradually.With the quickening growth rate of identical separating time points,the morphology changes from small and short to elongated ones.On the other hand,there is no relationship between growth rate and morphology on different separating time points.4 The virulence of isolates has been enhanced significantly and stably genetic inherited since the eighth generation of 24 hours and the seventh generation of 48 hours.