| Newly weaned rabbits are susceptible to intestinal diseases. Antibiotics substitutes such as probiotics will be widely used to prevent pathogen injection in rabbit farming due to the well-known disadvantages of antibiotics. Lactic acid bacteria(LAB) are the main probiotics for single stomach animals like swine and chiken, but they are not the prominent ones in rabbit’s gut. It needs further study to determine whether LAB can be developed as rabbit’s probiotics. The effects of LAB can be direct or indirect through modulation of the endogenous flora or of the immune system. Toll-like receptors(TLRs) are part of the innate immune response to microbial antigens. Recent studies had provided evidence that the TLR 9 receptor recognizes bacterial DNA, in particular sequences containing unmethylated CpG dinucleotides. The present study was conducted to isolate Lactobacillus and Paneth cells from rabbit small intestine and then to explore the effects of Lactobacillus genomic DNA on intestinal immune.The 5-day-old New Zealand white rabbits have been administered orally with L.casei for 8 days, then the development of their intestines and the secretion of their Paneth cells were detected. In vitro studies, isolated crypts from rabbit’s small intestine were stimulated with both bacterial DNA and specific short sequences for 5 hours at 37℃, and the expression of TLR 9, lysozyme, α-defensins, TNF-α, INF-β and IL-6 in crypts was detected.Results: 1) The bacterial isolates were identified as L.acidipiscis, L.casei, L.brevis and Enterococcus by 16 S rDNA sequencing. L.casei was chosen as the experimental bacteria for high GC content in its genomic DNA. 2) We separated and enriched intestinal villus and crypt under conditions with different chelating agent concentrations(5,10,15,20,25 mmol/L EDTA) and chelating temperatures(4℃,25℃). Our results showed that all of the collected villus and crypt remained morphological integrity under different chelating separation conditions, and the genomic DNA and total RNA of the separated villus and crypt were intact. Best separation effort was observed under 4℃ and 10 mmol/L EDTA in our experiment. The relative expression levels of lysozyme and α-defensin genes of enriched crypt were significantly higher than those of the separated villus(P<0.01). In conclusion, the improved method was suitable for separating rabbit intestinal villus and crypt cells effectively. 3) Compared with the control group, oral administering with L.casei significantly increased the proportion of Lactobacillus to total bacteria in intestine(P<0.01), and reduced that of Escherichia coli to total bacteria(P<0.01), and also enhanced the expression of TLR 9, lyszome and α-defensin in duodenum and jejunum(P<0.05), but not in ileum.More degranulated Paneth cells degranulation were as observed in duodenum and jejunum sections from rabbit orally treated with L.casei(P<0.01). 4) The genomic DNA of L.casei without digestion by enzyme had no significant effect on crypt cells. 5) In the present study, we found that CpG 2007 is a potent inducer of secretion and cytokine production for rabbit’s intestinal Paneth cell. The digested genomic DNA of L.casei by Sau3A1 showed stimulatory effect on the expression of TLR 9 receptor and α-defensin mRNA, but no significant effect on that of lysozyme, interleukin(IL)-6, tumour necrosis factor alpha(TNF-α) and interferon gamma(IFN-β) mRNA.Therefore, L.casei improved the level of intestinal immune in rabbit. Small fragments from the enzymolysis of bacterial genome DNA can be recognized by the host and activated the innate immune. Our study provided a certain theory basis for the reasonable use of lactic acid bacteria as a probiotic in rabbit farming. |
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