The flagella of F18ab Escherichia coli is a virulence factor that contributes to infection in a IPEC-J2 cell model in vitro

Bacterial flagella contribute to pathogen virulence; however, the role of flagella in the pathogenesis of F18ab E. coli-mediated swine edema disease (ED) is not currently known. We therefore evaluated the role of flagella in F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production using an in vitro cell infection model approach with gene-deletion mutant and complemented bacterial strains. We demonstrated that the flagellin-deficient fliC mutant had a marked decrease in the ability to adhere to and invade porcine epithelial IPEC-J2 cells. Surprisingly, there was no difference in adhesion between the F18 fimbriae-deficient ΔfedA mutant and its parent strain. In addition, both the ΔfedA and double ΔfliCΔfedA mutants exhibited an increased ability to invade IPEC-J2 cells compared to the wild-type strain, although this may be due to increased expression of other adhesins following the loss of F18ab fimbriae and flagella. Compared to the wild-type strain, the ΔfliC mutant showed significantly reduced ability to form biofilm, whereas the ΔfedA mutant increased biofilm formation. Although ΔfliC, ΔfedA, and ΔfliCΔfedA mutants had a reduced ability to stimulate IL-8 production from infected Caco-2 cells, the ΔfliC mutant impaired this ability to a greater extent than the ΔfedA mutant. The results from this study clearly demonstrate that flagella are required for efficient F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production in vitro.     

Flagellin and F4 fimbriae have opposite effects on biofilm formation and quorum sensing in F4ac+ enterotoxigenic Escherichia coli

Bacteria that form biofilms are often highly resistant to antibiotics and are capable of evading the host immune system. To evaluate the role of flagellin and F4 fimbriae on biofilm formation by enterotoxigenic Escherichia coli (ETEC), we deleted the fliC (encoding the major flagellin protein) and/or the faeG (encoding the major subunit of F4 fimbriae) genes from ETEC C83902. Biofilm formation was reduced in the fliC mutant but increased in the faeG mutant, as compared with the wild-type strain. The expression of AI-2 quorum sensing associated genes was regulated in the fliC and faeG mutants, consistent with the biofilm formation of these strains. But, deleting fliC and/or faeG also inhibited AI-2 quorum sensing activity.     

Enteric-coated pellets of F4 fimbriae for oral vaccination of suckling piglets against enterotoxigenic Escherichia coli infections

To prevent enterotoxigenic Escherichia coli (ETEC) induced postweaning diarrhoea, the piglet needs an active mucosal immunity at the moment of weaning. In the present study, the feasibility of oral vaccination of suckling piglets against F4+ETEC infection with F4 fimbriae was studied. Furthermore, oral vaccination with enteric-coated pellets of F4 fimbriae was compared to vaccination with F4 fimbriae in solution. Therefore, piglets were orally administered 1mg F4 fimbriae in pellets or in solution during three successive days at the age of 7 and 21 days, whereas control piglets were not vaccinated. Five days postweaning (33 days of age), all animals were orally challenged with F4+ETEC. Despite the induction of an immune response upon oral administration of both F4 fimbriae in pellets as in solution, the colonisation of the small intestine by F4+ETEC upon oral challenge could not be prevented. However, a marginal but significant reduction in F4+ E. coli faecal excretion was found in the piglets vaccinated with F4 fimbriae in pellets, indicating that the use of an enteric-coat which protects the F4 fimbriae against inactivation by milk factors and degradation by enzymes and bile improves vaccination.     

Identification of infant and adult swine susceptible to enterotoxigenic Escherichia coli by detection of receptors for F4(K88)ac fimbriae in brush borders or feces.

We attempted to determine F4(K88)-adhesive and non-adhesive phenotypes of infant (neonatal <3 day old and weaned <4 week old pigs) and adult (>6 month old) swine by ELISA using immobilized F4(K88)ac fimbrial antigen or whole F4(K88) + E. coli cells (strains M1823 and 1476) and isolated small intestinal brush borders or easily-obtainable fecal samples from the same animals. Nineteen of 22 neonates (86%), 17 of 20 weaners (85%), and 26 of 39 adults (67%) were classified identically as F4(K88) receptor-positive or negative by the ELISA. The ELISA with feces from adult swine was found to be almost equally specific (87%) as that with feces from neonatal (90%) and weaned (91%) pigs. However, the sensitivity of the assay was low (38%), indicating that fecal samples from adults contained less receptor-material than necessary for comparable phenotyping. The receptor-positive brush borders from neonates and weaners reacted significantly better (P < 0.02, < 0.001 respectively) with purified F4(K88) antigen than did those from adults. There was good agreement between the average ELISA values for feces from infant and adult swine regardless the source of coating antigen applied. With this assay we can determine F4(K88) phenotypes of infant swine using easily-collected fecal samples rather than isolated brush borders. It was also concluded that tested feces is not an acceptable alternate source of the receptor-material to brush borders from F4(K88)-susceptible adult swine.     

Saccharomyces cerevisiae decreases inflammatory responses induced by F4+ enterotoxigenic Escherichia coli in porcine intestinal epithelial cells

Probiotic yeasts may provide protection against intestinal inflammation induced by enteric pathogens. In piglets, infection with F4+ enterotoxigenic Escherichia coli (ETEC) leads to inflammation, diarrhea and intestinal damage. In this study, we investigated whether the yeast strains Saccharomyces cerevisiae (Sc, strain CNCM I-3856) and S. cerevisiae variety boulardii (Sb, strain CNCM I-3799) decreased the expression of pro-inflammatory cytokines and chemokines in intestinal epithelial IPI-2I cells cultured with F4+ ETEC. Results showed that viable Sc inhibited the ETEC-induced TNF-α gene expression whereas Sb did not. In contrast, killed Sc failed to inhibit the expression of pro-inflammatory genes. This inhibition was dependent on secreted soluble factors. Sc culture supernatant decreased the TNF-α, IL-1α, IL-6, IL-8, CXCL2 and CCL20 ETEC-induced mRNA. Furthermore, Sc culture supernatant filtrated fraction < 10 kDa displayed the same effects excepted for TNF-α. Thus, our results extended to Sc (strain CNCM I-3856) the inhibitory effects of some probiotic yeast strains onto inflammation.     

Seroprevalence of F4+ enterotoxigenic Escherichia coli in regions with different pig farm densities.

Sera of young sows from 135 closed Belgian pig breeding farms were examined for the presence of serum antibodies specific for the F4 fimbrial antigen of enterotoxigenic Escherichia coli (ETEC). Since 80% of all pig farms in Belgium are located in the provinces West-Vlaanderen (44%, approximately 18 farms/10 km2), Oost-Vlaanderen (20%, approximately 9 farms/10 km2), Antwerpen (10%, approximately 5 farms/10 km2) and Vlaams-Brabant (6%, approximately 3 farms/10 km2), the farms examined were randomly selected in these four regions. On 68% of all tested farms, sows were not vaccinated against enteric colibacillosis. In general, 65% of these non-vaccinated farms were F4-seropositive (mean optical density [OD405] >0.5) of which 38% were weakly (mean OD405 = 0.5-1.0), 21% moderately (mean OD405 = 1.0-2.0) and 6% strongly positive (mean OD405 > or =2.0). These percentages showed major differences between the four provinces. In Vlaams-Brabant, only 31% of non-vaccinated farms were seropositive, of which most were regarded as weakly positive. In Antwerpen, Oost- and West-Vlaanderen however, this percentage was clearly higher and respectively 68, 71, and 79% of non-vaccinated farms were seropositive, of which most were weakly positive. These observations indicate that F4+ ETEC is widely spread and highly prevalent on non-vaccinated pig breeding farms in Belgium. Moreover, an increasing F4-seropositivity with increasing pig farm density suggests a possible influence of the swine density on the prevalence of F4+ ETEC infections.     

Active oral immunization of suckling piglets to prevent colonization after weaning by enterotoxigenic Escherichia coli with fimbriae F18

Immunoprophylaxis of porcine oedema disease and post-weaning diarrhoea caused by strains of Escherichia coli expressing fimbriae F18 is an unsolved problem. The study was designed to examine whether vaccination with a live F18ac vaccine of unweaned pigs born to sows with F18ac antibody in the colostrum requires preformed fimbriae in the vaccine, and whether protection against the heterologous fimbrial variant F18ab is induced as well. Genetically susceptible pigs were vaccinated orally on three consecutive days, beginning 10 days before weaning with 10(11) CFU of an F18ac culture. Challenge with a dose of 10(7) CFU of E. coli F18 on three consecutive days was initiated 9 or 11 days after weaning. Eighteen pigs given the fimbriated F18ac vaccine and challenged with a strain of the homologous fimbrial variant were protected against colonization; mean faecal viable counts of the challenge strain were >3 log10 lower than those from the 17 non-vaccinated control pigs. The vaccinated pigs developed a significant rise of F18ac IgA serum antibodies. The 23 pigs which had received the non-fimbriated vaccine showed no significant protection and exhibited much lower serum F18ac IgA ELISA reactivities. Eighteen pigs vaccinated with the fimbriated F18ac and challenged with an F18ab strain had faecal viable counts nearly as high as those from 16 non-vaccinated control pigs. It is concluded that only oral vaccines having preformed fimbriae induce protection limited to the homologous fimbrial variant.     

The search for the gene mutations underlying enterotoxigenic Escherichia coli F4ab/ac susceptibility in pigs: a review

Diarrhoea due to enterotoxigenic Escherichia coli with fimbriae F4 (ETEC-F4) is an important problem in neonatal and just weaned piglets and hence for the pig farming industry. There is substantial evidence for a genetic basis for susceptibility to ETEC-F4 since not all piglets suffer from diarrhoea after an ETEC-F4 infection. It is assumed that the wild boar was originally ETEC-F4 resistant and that susceptibility towards ETEC arose after domestication. There are different phenotypes in the pig determined by which of the three existing F4 variants (F4ab, F4ac or F4ad) they are susceptible or resistant for. This suggests that several F4 receptors exist, expressed individually or in combination with each other on the brush border of the piglet’s small intestine. As such, the mucin-type glycoproteins (IMTGP) are described as F4ab/ac receptors, while the intestinal neutral glycospingolipid (IGLad) is proposed as an F4ad receptor. GP74 is a putative F4ab receptor. However, the specific genes that encode for the susceptibility are not yet known. In the past decades, linkage analyses revealed that the loci encoding for the receptor(s) for the two most frequent variants F4ab and F4ac were mapped to the 13^th chromosome of the pig (Sus scrofa 13, SSC13). After fine mapping, the region of interest was mapped between two microsatellite markers, Sw207 and S0075, and interesting candidate genes surfaced. Numerous SNP analyses and a few expression studies on the three MUC-genes (MUC4, MUC13 and MUC20) and the transferrin receptor gene (TFRC) as well as on some other positional candidate genes have been performed in order to find the causative mutation for the ETEC-F4ab/ac receptor(s). However, until today, the exact mutation causing susceptibility to ETEC-F4 remains unknown.     

Levamisole synergizes experimental F4ac+ Escherichia coli oral vaccine in stimulating ileal Peyer's patch T cells in weaned pigs

Recent findings demonstrate that priming by levamisole of weaned pigs experimentally vaccinated against postweaning colibacillosis (PWC) contributes to immune protection from challenge-induced clinical disease through stimulation of the mesenteric lymph node cells that participate in cell-mediated immunity. With the objective of better understanding the mechanisms by which levamisole induces protective mucosal cell-mediated immune response to vaccination against PWC, it was tested whether the drug synergizes experimental F4ac+ Escherichia coli oral vaccine in stimulating T cells also in the jejunal lamina propria (JLP) and ileal Peyer's patch (IPP) upon virulent challenge. Commercial crossbred pigs weaned at 4 weeks were allocated into two equal groups. The experimental group was i.m. primed with levamisole at an immunostimulatory dose of 2.5 mg/kg once daily, for 3 consecutive days, and controls received saline. Both groups were vaccinated orally with the vaccinal E. coli strain on day 0 and challenged with the virulent E. coli strain 7 days later. All pigs were killed on postchallenge day 6. The results determined by immunophenotyping of isolated cells indicate that priming by levamisole of the vaccinated weaned pigs selectively recruited and activated T cells in the IPP, a lymphoid organ-generating B lymphocytes. The pig IPP is normally populated with up to 5% of CD3+ T cells and CD6 is an activation antigen expressed exclusively by T cells in swine. Therefore, a significantly higher number of CD3+ (P < 0.01) and CD6+ (P < 0.001) cells observed within the IPP follicles of the primed-vaccinated vs. unprimed-vaccinated challenge-infected pigs suggest enhanced T cell-mediated immunity in this B-cell compartment induced by the potentiating action of the drug and vaccine. The ability of levamisole to influence interaction between activated T cells and B cells in the IPP of primed-vaccinated weaned pigs, and the possibility that this interaction plays a role in regulating B-cell maturation within the IPP follicles, are discussed.     

Levamisole synergizes proliferation of intestinal IgA+ cells in weaned pigs immunized with vaccine candidate F4ac+ nonenterotoxigenic Escherichia coli strain

Levamisole (2, 3, 5, 6-tetrahydro-6-phenylimidazole 2,1-b thiazole) is a well-known nonspecific stimulator of host defence mechanisms. In previous investigations, we have found that levamisole acts on cell-mediated immunity in challenge-induced porcine postweaning colibacillosis (PWC). We assume that levamisole could also act synergistically on humoural immune response when applied as an adjuvant with vaccine candidate strains for oral immunization of weaned pigs against PWC. The influence of levamisole in combination with experimental F4ac⁺ nonenterotoxigenic Escherichia coli (non-ETEC) vacinal strain on proliferation of IgA⁺ cells was examined in 4-week-old weaned pigs experimentally infected with ETEC. We have performed identification and morphometric quantification of the plasma cell phenotype within jejunal/ileal mucosa. Plasma cells were identified by immunohistochemistry with monoclonal anti-IgA antibodies and quantifying by use of digital image analysis. Quantification of IgA⁺ cells from levamisole-primed vaccinated and challenge-infected weaned pigs showed significantly increased number ( P  < 0.05 for both jejunum and ileum) compared with those observed in unprimed vaccinated/challenge-infected controls. It is suggested from these results that levamisole may contribute in initiation of local humoural immune response to enteric pathogens, such as enterotoxigenic E. coli .     

Screening of pigs resistant to F4 enterotoxigenic Escherichia coli (ETEC) infection

The present study analysed quantitatively the mucin 4 polymorphism for determining the F4ac/ab receptor status of a total of 63 pigs by comparing it with the in vitro villous adhesion assay. The probability of a susceptible genotype for the mucin 4 increases significantly with increasing F4ab or F4ac ETEC adhesion per 250 microm villi (P=0.029 for F4ab, P=0.030 for F4ac), with the odds ratio for each unit increase of F4ab or F4ac equal to, respectively, 1.036 (95% CI [1.004-1.069]) and 1.018 (95% CI [1.002-1.034]). In the phenotypic in vitro villous adhesion test, a cut-off value of 5 bacteria was chosen as a criteria for the distinction between an F4R positive and F4R negative pig. The sensitivity and specificity for the in vitro villous adhesion test, with the genotyping test for mucin 4 as golden standard, is 100% and 24%, respectively, for F4ab as well as F4ac. Absence of adhesion of F4ac and F4ab ETEC to the villous brush borders was not associated with genotypic resistance suggesting that there is at least one other receptor for F4ab/ac Escherichia coli. As a consequence, not only mucin 4 gene polymorphism but also expression of these other receptor(s) has to be included in a screening assay for F4ac/ab receptor negative pigs.     

Replicon typing of F18 fimbriae encoding plasmids of enterotoxigenic and verotoxigenic Escherichia coli strains from porcine postweaning diarrhoea and oedema disease

The presence of fimbrial adhesin F18 is frequently found in enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) strains responsible for diarrhoea and oedema disease of weaned pigs. The F18 adhesin occurs in two antigenic variants: F18ab is characteristic of VTEC while F18ac is more typical for ETEC. F18 encoding plasmids of 17 phenotypically characterized porcine E. coli isolates (10 ETEC, 6 VTEC and 1 ETEC/VTEC) were tested with a DNA probe for F18 fimbrial adhesin and with replicon probes for the RepFIa, RepFIb and for the RepFIc family of basic replicons. In all the cases, the F18 probe hybridized to only one plasmid band of size higher than 42MDa. All F18 plasmids were determined to be unireplicon plasmids belonging to the RepFIc replicon family of the F incompatibility complex. There was no difference between F18ac plasmids of ETEC and F18ab plasmids of VTEC strains in terms of replicon type or subtype. However, the size of F18ab plasmids of the VTEC strains varied between 42 and 98MDa, in contrast to F18ac plasmids of ETEC strains (constantly approximately 98MDa).     

Purification and characterization of the fimbria F18ac (2134P) isolated from enterotoxigenic Escherichia coli (ETEC)

The adhesin F18ac purified on Sepharose CL 4B column chromatography and SDS-PAGE stained with Coomassie Blue and Western blotting using specific anti-F18ac serum presented one band of approximately 17kDa. Gold immunolabeling revealed that the adhesin F18ac has a fimbrial structure on the bacterial surface. The first 27 amino acid residues of the N-terminal portion of the adhesin F18ac, showed 92.5% homology (25 amino acids) with the F107 (F18ab) fimbriae.     

Administration of probiotics influences F4 (K88)-positive enterotoxigenic Escherichia coli attachment and intestinal cytokine expression in weaned pigs

ABSTRACT: This study evaluated the effect of the probiotics Pediococcus acidilactici and Saccharomyces cerevisiae boulardii on the intestinal colonization of O149 enterotoxigenic Escherichia coli harbouring the F4 (K88) fimbriae (ETEC F4) and on the expression of ileal cytokines in weaned pigs. At birth, different litters of pigs were randomly assigned to one of the following treatments: 1) control without antibiotics or probiotics (CTRL); 2) reference group in which chlortetracycline and tiamulin were added to weanling feed (ATB); 3) P. acidilactici; 4) S. cerevisiae boulardii; or 5) P. acidilactici + S. cerevisiae boulardii. Probiotics were administered daily (1 × 109 CFU per pig) during the lactation period and after weaning (day 21). At 28 days of age, all pigs were orally challenged with an ETEC F4 strain, and a necropsy was performed 24 h later. Intestinal segments were collected to evaluate bacterial colonization in the small intestine and ileal cytokine expressions. Attachment of ETEC F4 to the intestinal mucosa was significantly reduced in pigs treated with P. acidilactici or S. cerevisiae boulardii in comparison with the ATB group (P = 0.01 and P = 0.03, respectively). In addition, proinflammatory cytokines, such as IL-6, were upregulated in ETEC F4 challenged pigs treated with P. acidilactici alone or in combination with S. cerevisiae boulardii compared with the CTRL group. In conclusion, the administration of P. acidilactici or S. cerevisiae boulardii was effective in reducing ETEC F4 attachment to the ileal mucosa, whereas the presence of P. acidilactici was required to modulate the expression of intestinal inflammatory cytokines in pigs challenged with ETEC F4.     

Experimental infection with Escherichia coli O149:F4ac in weaned piglets

The outcome of experimental intestinal infections with enterotoxigenic Escherichia coli (ETEC) is dependent on several factors. An important factor is adhesion of the challenge strain to the intestinal mucosa. The test for susceptibility towards ETEC adhesion has so far been made by an intestinal adhesion test made after slaughter of piglets. However, in an experimental infection study with the purpose to obtain diarrhoeic piglets, it would be an advantage to test for susceptibility prior to experimentation. The Mucin 4 gene on porcine chromosome 13 has been proposed as a candidate gene for the production of the specific ETEC F4ab/ac receptor, and a DNA marker-based test has been developed to allow genotyping for ETEC F4ab/ac resistance/susceptibility [J?rgensen, C.B., Cirera, S., Archibald, A.L., Anderson, L., Fredholm, M., Edfors-Lilja, I., 2004. Porcine polymorphisms and methods for detecting them. International application published under the patent cooperation treaty (PCT). PCT/DK2003/000807 or WO2004/048606-A2]. The aim of this study was to test an experimental model for ETEC O149:F4ac-induced diarrhoea in piglets, selected for susceptibility towards ETEC O149:F4ac adhesion prior to experimentation using a DNA marker-based test. Sixty-two healthy 25-32 days old recently weaned Danish crossbred piglets were used. All piglets were tested prior to experimentation for susceptibility or resistance towards ETEC O149:F4ac adhesion. Thirty-nine piglets, both susceptible and resistant, were oro-gastric intubated with 10(9)CFU of ETEC O149:F4ac and 23 age-matched piglets, both susceptible and resistant, were used as non-infected controls. Of susceptible piglets, challenged with ETEC O149:F4ac, 74% had ETEC O149:F4ac-associated diarrhoea first day after first challenge, which were significantly higher relatively to the resistant and challenged piglets where 20% had diarrhoea (p=0.04). This study suggests a model for experimental ETEC induced diarrhoea.     

Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine

Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) increases bacterial adherence to porcine enterocytes in vitro and enhances small intestinal colonization in swine. Heat-stable enterotoxin-b (STb) is not known to affect colonization; however, through an induction of net fluid accumulation it might reduce bacterial adherence. The relationship between fluid accumulation and bacterial adherence in jejunal loops inoculated with ETEC strains that produce LT, STb, both, or neither toxin was studied. Ligated jejunal loops were constructed in weaned Yorkshire pigs in two independent experiments (Exp. 1, n = 5, 8-week-old; Exp. 2, n = 6, 6–8-week-old). Each pig was inoculated with six F4ac⁺ E. coli strains: (1) LT⁺, STb⁺ parent (WAM2317); (2) STb^? (ΔestB) mutant (MUN297); (3) MUN297 complemented with STb (MUN298); (4) LT^? STb^? (ΔeltAB ΔestB) mutant (MUN300); (5) MUN300 complemented with LT (MUN301); and (6) 1836-2 (non-enterotoxigenic, wild-type). Pigs were confirmed to be K88 (F4)ab/ac receptor-positive in Exp. 2 by testing for intestinal mucin-type glycoproteins and inferred to be receptor-positive in both Exp. 1 and 2 based on histopathologic evidence of bacterial adherence. Strains that produced STb induced marked fluid accumulation with the response (ml/cm) to WAM2317 and MUN298 significantly greater than that to the other strains (P < 0.0001). Conversely, bacterial adherence scores based on immunohistochemistry and CFU/g of washed mucosa were both lowest in the strains that expressed STb and highest in those that did not. For the two experiments combined, the Pearson correlation coefficient (R) between fluid volume (ml/cm) and log CFU per gram was ?0.57021 (P < 0.0001); R² = 0.3521 (n = 197). These results support the hypothesis that enterotoxin-induced fluid accumulation flushes progeny organisms into the lumen of the bowel, thereby increasing the likelihood of fecal shedding and transmission of the pathogen to new hosts.     

Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoea

Oral administration of polyclonal antibodies directed against enterotoxigenic Escherichia coli (ETEC) F4 fimbriae is used to protect against piglet post-weaning diarrhoea. For cost reasons, we aim to replace these polyclonal antibodies by recombinant llama single-domain antibody fragments (VHHs) that can be produced efficiently in microorganisms. Six F4 fimbriae specific VHHs were isolated. The VHH that was produced at the highest level by yeast, K609, was further analysed. 3.8 mg/L K609 inhibited 90% of bacterial attachment to intestinal brush borders in vitro. Perfusion of a jejunal segment with at least 4 mg/L K609 reduced the ETEC-induced fluid loss, but only to 30%. Preventive administration of a high K609 dose (150 mg/(piglet day)) to piglets that were challenge infected with ETEC resulted in less severe diarrhoea only at 4 and 5 days post-infection, but did not improve average daily weight gain, ETEC shedding and piglet survival. Thus, we have shown that an antibody fragment that effectively inhibited in vitro ETEC adhesion to intestinal brush borders poorly protected piglets against experimental ETEC infection.     

Effect of mucin 4 allele on susceptibility to experimental infection with enterotoxigenic F4 Escherichia coli in pigs fed experimental diets

Background: This study investigated the validity of the DNA-marker based test to determine susceptibility to ETECF4 diarrhoea by comparing the results of two DNA sequencing techniques in weaner pigs following experimental infection with F4 enterotoxigenic Escherichia coli(ETEC-F4). The effects of diet and genetic susceptibility were assessed by measuring the incidence of piglet post-weaning diarrhoea(PWD), faecal E. coli shedding and the diarrhoea index.Results: A DNA marker-based test targeting the mucin 4 gene(MUC4) that encodes F4 fimbria receptor identified pigs as either fully susceptible(SS), partially or mildly susceptible(SR), and resistant(RR) to developing ETEC-F4 diarrhoea. To further analyse this, DNA sequencing was undertaken, and a significantly higher proportion of C nucleotides was observed for RR and SR at the Xba I cleavage site genotypes when compared to SS. However, no significant difference was found between SR and RR genotypes. Therefore, results obtained from Sanger sequencing retrospectively allocated pigs into a resistant genotype(MUC4–), in the case of a C nucleotide, and a susceptible genotype(MUC4+), in the case of a G nucleotide, at the single nucleotide polymorphism site. A total of 72 weaner pigs(age ～ 21 days), weighing 6.1 ± 1.2 kg(mean ± SEM), were fed 3 different diets:(i) positive control(PC) group supplemented with 3 g/kg zinc oxide(Zn O),(ii) negative control(NC) group(no Zn O or HAMSA),and(iii) a diet containing a 50 g/kg high-amylose maize starch product(HAMSA) esterified with acetate. At days five and six after weaning, all pigs were orally infected with ETEC(serotype O149:F4; toxins LT1, ST1, ST2 and EAST). The percentage of pigs that developed diarrhoea following infection was higher(P = 0.05) in MUC4+ pigs compared to MUC4– pigs(50% vs. 26.8%, respectively). Furthermore, pigs fed Zn O had less ETEC-F4 diarrhoea(P = 0.009) than pigs fed other diets, however faecal shedding of ETEC was similar(P 0.05) between diets.Conclusion: These results confirm that MUC4+ pigs have a higher prevalence of ETEC-F4 diarrhoea following exposure, and that pigs fed Zn O, irrespective of MUC4 status, have reduced ETEC-F4 diarrhoea. Additionally,sequencing or quantifying the single nucleotide polymorphism distribution at the Xba I cleavage site may be more reliable in identifying genotypic susceptibility when compared to traditional methods.     

干酪乳杆菌对产肠毒素大肠杆菌粘附Caco-2细胞抑制作用的研究

为研究干酪乳杆菌(L.casei)对人结肠癌Caco-2细胞的粘附特点及抑制产肠毒素大肠杆菌K88(ETEC K88)粘附Caco-2细胞的能力,本研究将不同浓度的L.casei和ETEC K88与分化完全的Caco-2细胞共培养2 h和4 h后,采用平板计数法和革兰染色法计算每个细胞上粘附菌的个数,评价L.casei的粘附特点及其抑制ETEC K88对Caco-2细胞的粘附能力,并分析其影响因素。结果表明,L.casei与ETEC K88均可以粘附至Caco-2细胞表面,并且随菌液浓度的升高和培养时间的延长,粘附的数量显著增加(p0.05)。L.casei浓度越高,抑制ETEC K88粘附至细胞表面的能力越强,并与菌液添加顺序有关,但培养时间对其影响不显著(p0.05)。本研究为L.casei抑菌作用机制的研究提供了实验依据。