The Establishment And Application Of Caenorhabditis Elegans As Animal Model For Study Probitics Bacteria

The study concerning effection and mechanism of Lactobacillus has been restricted by complicated intestinal microflora of mammal animals and expensive cost on germ-free animals. Caenorhabditis elegans has been used as a laboratory animal model for the fact that there is a broad overlap between mammals and C. elegans about infection mechanism, immune reaction and so on. In this study, Salmonella used for pathogen and Lactobacillus used for probiotic. Caenorhabditis elegans has been used for estabilished a laboratory animal model for Conduct research on probiotic.The C.elegans strain SS104 has been hatched firstly by fluild incubation method, and proper dose of S. typhimurium and Llactobacillus has been confirmed. We have evaluated 17 Lactobacillus isolates from chicken and pig intestine for their ability to protect the worms from Salmonella-induced death in vitro; When tested on C. elegans infected with S. typhimurium DT104, the isolates were, however, differentiated in their ability to protect the nematode from Salmonella-induced death. Two Lactobacillus isolates S64 and CL11 (S64 with full, but CL11 with no protection) were further studied. The study on difference culture fractions (the extracellular culture fluid, live and heat-killed cells) of isolates CL11 and S64 has been designed; We observed the effects of Lactobacillus isolates on performance , diarrhea incidence, immune activities and intestinal microflora of wean piglets and layer chicken in order to evaluate similarity between livestock and C.elegans.The study consists of four parts:1 Establishment of the assay with C. elegans used as an animal moder for study probioticsSynchronizated L4 larvae has been hatched, resuspension in 24-well titre plates with each well containing 2 ml of S medium and 10– 15 worms, and incubated at 25°C during the assay. Each treatment had four wells. To explore the experimental condition of C.elegans used as animal model for evaluate the effect of a Lactobacillus isolate on protecting the nematode from Salmonella-induced death. The result showed : the fluid incubation method of C.elegans was more easy to operate; C.elegans was infected and died with S. typhimurium at 10⁷cfu/ml,10⁸cfu/ml and 10⁹cfu/ml, however, all the worms died in 6^th days at 10⁹cfu/ml;The survival rate (87.9%) of Lactobacillus-treated worms at 108cfu/ml was higher than that of C. elegans fed E. coli OP50 (82.8%) only on 10^th day, but survival rate of C.elegans was 63.2% at 10⁹cfu/ml; The protective rate of Lactobacillus for C.elegans was 50.3% . meanwhile, the green fluorescence was emitted by the GFP- Salmonella of Lactobacillus-treated worms was more diminish than positive control in the digestive tract of the worms.2 Presreening of Lactobacillus isolates using a laboratory animal model of Caenorhabditis elegans17 Lactobacillus isolates has been confirmed by 16S rRNA analysis method. The spot-on-the-lawn assay demonstrated that all 17 tested Lactobacillus isolates were inhibitive to S. typhimurium . The size of growth inhibition haloes ranged from 19 to 25 mm in diameter and the extracellular culture fluid (ECF) recovered from tested Lactobacillus cultures all had a similar degree of inhibition towards S. typhimurium; When tested on C. elegans infected with S. typhimurium, the isolates were, however, differentiated in their ability to protect the nematode from Salmonella-induced death. On the last day (9^th) of the assays, the protective rate of Lactobacillus ranged from -2.6% to -56%. Among them, five isolates (CL9, L3, LB1, LB4, and S64) provided maximum protection(59%,53.2%,51.1%,58.7% and 55.7% respectively ) (P﹤0.05) to the infected worms that had a similar level of longevity compared to the negative control. The protective rate of two isolates CL10 and CL11 was -3.2% and -6.2%(P﹤0.01) and demonstrated negative effective on C.elegans . Our observations suggest that C. elegans can be used as an animal model for pre-screening probiotics.3 Application research on Caenorhabditis elegans as an animal model for study probaotics——Effection of Lactobacillus isolates S64 and CL11 screened by C.elegans To explore possible mechanisms underlying the protection offered by Lactobacillus different culture fractions, including the ECF, live and heat-killed cells, of Lactobacillus isolate (S64 and CL11) were investigated for their effect on the survival rate C. elegans infected with Salmonella and the colonization of the pathogen in the digestive tract of the nematode. The result demonstrated: (1)On the 9th day in the assay, the survival rate of C.elegans treated with lactobacillus isolate S64 and CL11 live cells was 72.2% and 25.7%(P<0.05), the protetive rate was 51.7% and 2.2% respectively; The survival rate of C.elegans treated with lactobacillus isolate S64 and CL11 heat-killed cells was 57.4% and 25.8%(P<0.05),the protetive rate was 33.9% and 2.3% respectively; The survival rate of C.elegans treated with ECF of S64 and CL11 was 36.7% and 35.8%(P>0.05),the protetive rate was 13.2% and 12.3% respectively. (2) On the 8^th day in the assay , the amount of Lactobacillus in the digestive tract treated with live Lactobacillus isolate S64 was increase distinctly compare to that of treated with live isolate CL11(P<0.05); The amount of Salmonella treated with live and heat-killed isolate S64 was significantly reduced than that of treated with isolate CL11 and positive control(P<0.05); The amount of Salmonella treated with ECF of isolate S64 and CL11 was at similar degree(P>0.05); Meanwhile, the green fluorescence was emitted by the GFP-salmonella of live isolate S64-treated worms was significantly diminished than positive control and isolate CL11-treated worms.4 Application research on Caenorhabditis elegans as an animal model for study probaotics——Effection of Lactobacillus isolates screened by C.elegans on livestockTo explore the effction of effection of Lactobacillus isolates screened by C.elegans on livestock , the feeding Trial had beedn designed using wean piglet and chicken as animal modle.(1) 96 weaned piglets were divided into 8 groups randomly and fed them with ferment liquid feed (FLF) which fermented by Lactobacillus isolates S64,LB1,LB2,ALB2,L3,CL11,dry feed (control 1) and liquid feed (control 2, no lactobacillus) respectively. The result showed: The ratio of feed to meat was reduced 49%,47%,36% and 24%(P<0.05), diarrhea index was reduced 50.89%,64.29%,93.51% and 32.5%(P<0.05),Diarrhea incidence was dropped by 50.01%,81.82%,63.65% and 23.72% (P<0.05), when fed them with FLF fermented by Lactobacillus isolates ALB2,S64,LB1 and L3 respectively; The ratio of feed to meat was increased 4% and12% (P < 0.05),diarrhea index was increased 15% and 27.27%(P<0.05),diarrhea incidence increased 21.78% and 29.95%(P<0.05), when fed them with FLF fermented by Lactobacillus isolates CL11 and LB2 respectively; Lactobacillus isolates LB1 was beneficial to the balance of gut flora than Lactobacillus isolates LB2.(2)A total of 200 newly hatched Layer Chicken were randomly allotted into 4 dietary treatments fed them with Lactobacillus isolates S64, L3 and CL11. On 35d in the assay, the hemagglutination inhibition antibody potency of Newcastle disease vaccine immunized of Lactobaccilus isolates S64 and L3 increased distinctly compare to negative control and isolate CL11(P<0.05); The amounts of Lactobaccilus in the Cecal contents significantly increased (P<0.05), and the amounts of E.coli and and Salmonella significantly reduced (P<0.05) when fed them with Lactobacillus isolate S64 and L3. Immune organ index of layer chicken when fed them with Lactobacillus isolate S64 improved significantly(P<0.05). The data of the treatment fed with Lactobacillus isolate CL11 was similar to negative control(P>0.05).The intestinal flora of the chickens fed Lactobacillus isolates S64 was changed clearly using ERIC-PCR based fingerprintsThese data demonstrated Lactobacillus isolates S64 and LB1 which screened by C.elegans was beneficial to production performance of wean piglets and to immunologic function of chickens. The result showed that the effection of Lactobacillus is similar between C.elegans and livestocks, and C.elegans can be used for study probiotic as a laboratory animal model.