The Effect Of Lactobacillus And Escheirchia Coli On Cryptospoirdium Parvum Infecting BALB/c Mice

Now people focus on studying the expression of intestinal antimicrobial peptideduring the interaction between bacteria and intestinal epithelial cells,as well as theeffection of parasite on the normal intestinal flora.However, only several reports focuson the influence of the bacteria on the process of parasites infecting mice, mainly onthe protection of probiotics,such as Lactobacillus reuteri，Lactobacillus casei andLactobacillus acidophilus,on protozoan infections.Also, the effect of pathogenicEscherichia coli and Lactobacillus bulgaricus on the process of Cryptosporidiuminfecting mice have not been reported after they colonized alone or together in theintestine.Therefore, in order to study the effect of pathogenic Escherichia coli andLactobacillus bulgaricus on the process of Cryptosporidium infecting BALB/c miceafter they colonized alone or together in the intestine，as well as the mechanism ofintestinal immune response, this experiment detected the colonization of pathogenicEscherichia coli and Lactobacillus bulgaricus at first, then infected BALB/c micewith Cryptosporidium parvum and monitored oocysts excreting patterns, intestinalcytokines, antimicrobial peptide in host defense response during the peak of oocystshedding.This research indicated the effect of pathogenic Escherichia coli andLactobacillus bulgaricus on the process of Cryptosporidium infecting BALB/c miceafter they colonized alone or together in the intestine and provided some theoreticalbasis for the mechanism of intestinal immune response.Detection of the colonization of Lactobacillus and E. coli in the mouse intestine50BALB/c mice of3weeks old were randomly divided into A, B, C, D and E group,representing the Cryptosporidium parvum group,the Lactobacillus group,the E. coligroup,the Lactobacillus and E. coli group and the blank group,respectively. Group Bwere continuously fed with10~8cfu Lactobacillus; group C with10~8cfu of E. coli,Group D with10~8cfu Lactobacillus and E. coli.A and E group were continuously fed with the same volume of PBS. We added5mg/L dexamethasone (DEX) to the sterilewater and fed the mice everyday, then counted the number of bacteria in the fecalsamples after a week.The results of the quantitative PCR show that compared withthe blank group, the number of Lactobacillus in Group B and Group D increasedsignificantly, indicating the successful colonization of Lactobacillus bulgaricus in theintestine. Similarly, in Group C and Group D, the number of E. coli increasedsignificantly, indicating the successful colonization of pathogenic E. coli in theintestine.The detection of oocysts excreting patterns and the expression of cytokineA, B, C and D groups of mice were infected orally with1mL PBS containing5×10~5Cryptosporidium parvum oocysts, and then observed the oocysts discharge law ineach group of mice. The results showed that mice in group B, C, and D excretedoocysts3d after infection, the volume of oocyst discharge reached the peak about11dafter infection，2d earlier than group A. Compared to group B, the volume of oocystdischarge in group C decreased significantly (p <0.05), and there were no significantdifference (p>0.05) in group D.11d after infection, mice in each group weresacrificed, collecting12cm small intestine from the ileocecal orifice.Then the smallintestine was divided into three equal parts.Took one part and collected thesupernatant after centrifugation. Then,the expression level of IL-2, IL-4, IL-12andIFN-γ were detected by ELISA. The results showed that compared with the blankgroup,the expression levels of IFN-γ in other groups were increased; the expressionlevel of IL-2and IL-4in Lactobacillus group, Lactobacillus and E. coli group andCryptosporidium parvum group increased while in E. coli group there was nosignificant changes; the expression levels of IL-12in Lactobacillus group,Lactobacillus and E. coli group and Cryptosporidium parvum group were downregulated, while in E. coli group there was no significant difference. Compared withthe Cryptosporidium parvum group,the expression level of IFN-γ in Lactobacillusgroup significantly increased (450.67±19.33pg/mL, vs.356.42±18.60pg/mL, p<0.01); the expression of IFN-γ in Lactobacillus and E. coli group significantlyincreased (409.36±20.10pg/mL vs356.42±18.60pg/mL, p <0.05);the expressionlevel of IFN-γ in E. coli group significantly decreased(p <0.01); the expression level of IL-12in Lactobacillus group significantly decreased (3.74±0.08pg/mL vs.4.23±0.14pg/mL, p <0.05); the expression level of IL-12in Lactobacillus and E. coli wassignificantly down regulated(3.68±0.13pg/mL vs.4.23±0.14pg/mL, p <0.05). Theresults indicated that the Thl-type cytokines, IFN-γ, IL-2and Th2-type cytokines IL-4played important roles in the immune response in BALB/c mice during theCryptosporidium parvum infection. The colonization of Lactobacillus bulgaricus canenhance the expression of IFN-γ, IL-2and IL4in the immune response, and thecolonization of pathogenic E. coli can inhibit the expression of IFN-gamma, IL-2andIL4.Detection of mouse intestinal defensins and lysozymeAntimicrobial peptides,mainly secreted by the intestinal epithelial cells, gobletcells and Paneth cells,are small molecular peptides with broad-spectrum antimicrobialactivity, playing an important role in the host defense against the invasion ofpathogenic microorganisms. Paneth cells, at the bottom of the midgut gland, cansynthesize and secrete lysozyme as well as defensins. In this study, we infected theBALB/c mice in each group with Cryptosporidium parvum,then collected thesmall intestine after11d,followed by RNA extraction and reverse transcription intocDNA. The results of Real-time PCR show that compared with blank control group,the expression level of intestinal lysozyme in Cryptosporidium parvum groupincreased (p <0.05), but the expression level of β defensin-1significantly decreased (p<0.01); the expression levels of β defensin-1and lysozyme in Lactobacillus groupwas not significantly different (p>0.05); the expression levels of β defensin-1andlysozyme in E. coli group was not significantly different (p>0.05); the expressionlevel of β defensin-1in Lactobacillus and E. coli group significantly increased (p<0.01);the expression level of lysozyme in blank group was not significantlydifferent. Compared with Cryptosporidium parvum group, the expression levels of βdefensin-1in Lactobacillus group and the E. coli group significantly increased, whilethe expression of lysozyme decreased. The results of H.E. staining show that thenumber of goblet cells in E. coli group, Cryptosporidium parvum and Lactobacillusand pathogenic E. coli group increased. After Lactobacillus bulgaricus andpathogenic E. coli colonizing alone or together in the intestine, the expression level of β-defensin is promoted, but the expression level of lysozyme is inhibited. The resultsof H.E. staining indicated that the goblet cells play an active role in the host defenseresponse.