| Diarrhoea, a major cause of mortality to newly weaned pigs, calves and humans is oftencaused by ETEC (Enterotoxigenic Escherichia E. coli). The swine industry has relied largelyon prophylactic use of antibiotics to control ETEC leading to a growing concern over thepractice due to the wide spread of antibiotic-resistance in zoonotic bacterial pathogens, whichposes a threat to public health. Thus, probiotics which is safe and healthy were widelyconsidered to be a potential alternative to dietary antibiotics. In addition, their beneficialeffects not only include reduction in colonization of animal intestines by pathogenic bacteria,but also contain improvement of animal performance, and enhancement of the animal immunesystem. Selection of LAB isolates with probiotic properties from a large number of bacteria iscritical in developing effective probiotics.In vitro model is convinient and fast but is quitedifferent from in-vivo environment while animal model is wide individual differences,expensive and time consuming. Thus, considering the application, cost, technology avaliable,C. elegans, a simple, short life-span, whole genomic sequencing, high conserved immunesystem, which is widely applying in pathology, neurology, cell biochemistry and drugreactions can be used as potential animal model to screen probiotics with specific advantage.In present study, an ETEC infecting C. elegans model and Lactobacillus protecting C.elegans from ETEC infecting screen model have been successfully eastablished. Furtherstudies were taken to figure out the protection machanism provided by the Lactobacillus LB1with best protection effect. At last, the C. elegans model and in vitro cell model were used tocompare their effect on screening the Lactobacillus and machanism studies. The results weredemonstrated as below:(1) In present study, an ETEC infecting C. elegans model and Lactobacillus protecting C.elegans from ETEC infecting screen model have been successfully established. The thirteenLactobacillus have quite different protection effect on worms, wherein the best protectionisolate LB1offered78%protection, the worst protection isolate CL11offered23%protection,and the rest offered23%-78%protections. Also the acid and bile salt resistant abilities weretested. Only LB1showed strong resistant abilities to both acid and bile salt while CL11andS20were sensitive to acid and bile salt and there is no relationship between acid tolerance andbile salt tolerance. In addition, LB1performed well at both protection C. elegans and acid,bile salt acid while S20, CL11showed no effect in both assays and other isolate showedmoderate effect in both assays. Therefore the best isolate LB1can be selected for furtherstudy.(2) The protection effect offered by probiotics is largely due to inhibit the colonizationand growth of pathogens. Therefore in present study the colonization of JG280and twoLactobacillus isolates (L. zeae LB1and L. casei CL11) in the intestine of C. elegans duringthe life-span assays was examined. The colonization level of JG280in the nematode intestineremained at approximately log104CFU/worm during the assay regardless of the absence orpresence of isolates LB1or CL11. Similarly, both Lactobacillus isolates had a similar level ofcolonization in the nematode intestine during the assay regardless of the absence or presence of JG280. These data indicate that intestinal colonization of JG280may not be the majorfactor causing the death of C. elegans and the Lactobacillus isolates had no effect on thecolonization of the nematode intestine by JG280. K88+strain JFF4is also a porcine isolatehaving F4/K88fimbriae but lacking enterotoxin genes of estA, estB, and elt. The JFF4strainhad no effect on C. elegans while the ETEC JG280can kill C. elegans in ten days, whichdemonstrated that the enterotoxin may play key role in ETEC JG280infection with worms.Thus three enterotoxins were cloned and the result showed that STa and STb clones havesimilar effect with ETEC JG280while LT clone only40%worms in10days. Thus wespeculated that the protection effect offered by Lactobacillus may relate to inhibitenterotoxins. The enterotoxin gene expressions were remarkable reduced when the C. eleganspretreated with Lactobacillus LB1. Also the death caused by enterotoxins clones wereinhibited by LB1. In summary, the protection offered by Lactobacillus may due to inhibitenterotoxins.(3) In the present study, we have investigated the host response of C. elegans to ETECinfection (strain JG280) and also the regulation of host signaling by Lactobacillus isolateswith or without a capacity to offer the protection. The results showed that not only selectedbut also viable not heat-killed Lactobacillus could induce immune responses of C. elegansagainst Enterotoxigenic Escherichia coli strain JG280infection. Also the life-span of nsy-1，sek-1，pmk-1mutants in MAPK pathway and daf-16mutant in Daf-2/Daf-16pathway weresignificant reduced infection with ETEC comparing to wild type, which indicated the MAPKand Daf-2/Daf-16pathway played key roles in C. elegans defence system against ETEC.Through the antimicrobial peptide genes expression in nsy-1and daf-16mutant showed thatabf-2，clec-85，lys-7，clec-60antimicrobial peptide genes were controlled by nsy-1and daf-16,spp-1was controlled by daf-16and abf-3was controlled by none of them. Compared with thewild-type, C. elegans mutants defective in age-1or dbl-1gene were more resistant to ETECinfection (longer lifespan), while the mutants defective in nsy-1or daf-16gene were lessresistant with a shorter lifespan. L. zeae LB1, an isolate showing the protective effect, wasable to further extend the lifespan of both age-1and dbl-1mutants infected with ETEC.However, no extension was observed with the nsy-1or daf-16mutant of C. elegans. Also asignificantly higher level in transcription of the antimicrobial peptide genes has been observedin the age-1and dbl-1mutants than in the nsy-1and daf-16mutants in response to ETECinfection. Interestingly, L. zeae LB1could further increase gene expression of theantimicrobial peptides in the age-1and dbl-1mutants, while it had no effect on the nsy-1anddaf-16mutants. Further studies with C. elegans mutants defective in antimicrobial peptidegenes have revealed that regulation in the production of antimicrobial peptides by L. zeae LB1played a key role in the protection of C. elegans from death caused by ETEC infection. Inconclusion, L. zeae LB1is able to regulate signalling of C. elegans through the MAPKpathway and DAF-16to control the production of antimicrobial peptides to combat ETECinfection.(4) Two assays with porcine intestinal epithelial IPEC-J2cells or Caenorhabditis elegansfor selecting effective probiotic candidates were compared. Both assays were based on measuring death of cells or worms caused by ETEC strain JG280. CL9，K16，K67，S33，LB1isolates provided maximum protection effect in both assays while CL10，CL11，CL12，K30，S20isolates offered minimal protection effect in both assays and the rest strains showdifferent protection effect in two models. The Lactobacillus protection effects were similar intwo models and it show significant relation R=0.61(P<0.0001，N=42). In general the fourteenisolates show similar protection effect in two models. The best isolate CL9was selected tocompare the effect on enterotoxins gene expression, enterotoxin clones and immuneregulation of host in both models. The results showed that isolate CL9could inhibitenterotoxins gene expression, reduce death caused by enterotoxin clones and regulate immunesystem in both C. elegans model and in vitro cell model. |
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