Features of enterotoxigenic Escherichia coli strains of porcine origin that express K88 and 987P fimbrial antigens

Escherichia coli strains of porcine origin express K88 and 987P pilus-antigens in vitro. This study reports their enterotoxin producing ability, serological features and plasmid content. The bipiliated strains were enterotoxigenic and all contained a large plasmid of uniform size.     

Pathogenicity of porcine enterotoxigenic Escherichia coli that do not express K88, K99, F41, or 987P adhesins.

Three-week-old weaned and colostrum-deprived neonatal (less than 1 day old) pigs were inoculated to determine the pathogenicity of 2 enterotoxigenic Escherichia coli isolates that do not express K88, K99, F41, or 987P adhesins (strains 2134 and 2171). Strains 2134 and 2171 were isolated from pigs that had diarrhea after weaning attributable to enterotoxigenic E coli infection. We found that both strains of E coli adhered in the ileum and caused diarrhea in pigs of both age groups. In control experiments, adherent bacteria were not seen in the ileum of pigs less than 1 day old or 3 weeks old that were noninoculated or inoculated with a nonpathogenic strain of E coli. These control pigs did not develop diarrhea. Antisera raised against strains 2134 and 2171 and absorbed with the autologous strain, grown at 18 C, were used for bacterial-agglutination and colony-immunoblot assays. Both absorbed antisera reacted with strains 2134 and 2171, but not with strains that express K99, F41, or 987P adhesins. A cross-reaction was observed with 2 wild-type K88 strains, but not with a K12 strain that expresses K88 pili. Indirect immunofluorescence with these absorbed antisera revealed adherent bacteria in frozen sections of ileum from pigs infected with either strain. We concluded that these strains are pathogenic and express a common surface antigen that may be a novel adhesin in E coli strains that cause diarrhea in weaned pigs.     

Determination of K88 antigens and enterotoxins of Escherichia coli strains isolated from Polish piglets with diarrhea by the use of enzyme-linked immunosorbent assays.

We studied 103 Escherichia coli strains isolated from suckling and weaned piglets with diarrhea using different ELISA tests. K88 fimbrial antigen was determined by the slide agglutination test and the ELISA inhibition method. LT and STa enterotoxins were tested directly in the microtiter plates using monoclonal antibodies. It was found that 56.3% strains possessed K88 antigen, all of which were of the K88ac type. There was 100% correlation between the slide agglutination and ELISA tests. Of the 103 strains tested 68.9% produced LT or STa or both toxins. LT-positive strains were the most common ones in both groups of piglets. All K88-positive strains were enterotoxigenic and elaborated LT (56 strains) or LT and STa (2 strains); STb production was not determined in this study. Our ELISA tests were easy to perform, specific and can be used for determination of K88 and enterotoxins in E. coli strains isolated from piglets.     

Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic Escherichia coli strains isolated from postweaning diarrhoea in pigs.

A novel common colonization factor was detected in enterotoxigenic strains of Escherichia coli isolated from intestinal contents of piglets affected with postweaning diarrhoea. This factor was antigenically distinct from the previously described K88, K99, F41, 987P, CFAI, CFAII and Att25 fimbrial antigens. E. coli strains possessing this factor adhered to the pig intestinal brush borders and one strain, used in experimental infection in weanlings, colonized the intestinal epithelium and induced diarrhoea. Examination of 212 toxigenic strains of E. coli isolated from weanlings revealed the presence of the novel common colonization factor in 83 strains, belonging to serogroups O25, O108, O138, O141, O147 and O157. The antigen K88 was detected in 47 strains belonging to serogroups O8, O141, O147 and O149.     

Real-time PCR for differentiation of F4 (K88) variants (F4ab, F4ac, F4ad) of enterotoxigenic Escherichiacoli from diarrhoeic piglets

A one-step real-time PCR using one set of oligonucleotide primers and three probes was developed for differentiation of F4 (K88) variants (F4ab, F4ac, F4ad) of enterotoxigenic Escherichiacoli (ETEC) from diarrhoeic pigs. The limits of detection of F4ab, F4ac and F4ad in broth dilution were 10(6), 10(5) and 10(4)colony forming units (CFU)/mL, respectively. In faecal samples spiked with E.coli, the limits of detection of F4ab, F4ac and F4ad were 10(6), 10(6) and 10(4)CFU/g faeces, respectively, without enrichment and 10(3), 10(2) and 10(2)CFU/g faeces following enrichment. In 42 ETEC field isolates from pigs in Korea encoding the F4 gene, all were identified as the F4ac variant.     

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.     

Phenotypic expression of K88 adhesin alone or simultaneously with K99 and/or F41 adhesins in the bovine enterotoxigenic Escherichia coli strain B41

F41-positive and F41-negative derivatives of bovine enterotoxigenic Escherichia coli strain B41 carrying K88 or K88 and K99 plasmids were investigated for stability and expression of genes for their fimbrial antigens. Either K88 plasmid alone or both K88 and K99 plasmids could be maintained in these strains though stability could depend on culture medium. K99 antigen could be detected in each strain bearing K99 plasmid. Clones that produced K88 antigen or clones that did not produce this antigen could be isolated from each strain, except from the strain that possessed K99 plasmid in the strain that did not possess the ability to produce F41 antigen. Strains possessing K88 plasmid in the strain able to produce F41 antigen produced clones expressing either both K88 and F41 antigens, (also F41 appeared strongly expressed in some clones) or clones that produced only F41 antigen or no antigen at all. Clones that produced only K88 antigen or others that did not produce this antigen could be produced from a strain bearing only K88 plasmid and that did not possess the ability to produce F41 antigen. None of these strains bearing K88 plasmid alone or additionally K99 plasmid produced mannose-resistant hemagglutination of horse or sheep erythrocytes at 20°C as found for K99 and F41 ETEC natural strains, respectively. These results suggested that the structures of pili when several genetic determinants were present simultaneously may not be identical to those of original strains. In this study, clones expressing either one, two or three adhesin bearing antigens could be obtained from the strain B41.     

Micro-encapsulated essential oils and organic acids combination improves intestinal barrier function,inflammatory responses and microbiota of weaned piglets challenged with enterotoxigenic Escherichia coli F4 (K88+)

This study evaluated the effects of micro-encapsulated (protected) organic acids (OA) and essential oils (EO) combination, P(OA + EO), and effects of a regular blend of free acids (FA) on the growth, immune responses, intestinal barrier and microbiota of weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC) F4 (K88⁺). A total of 30 crossbred (Duroc × Landrace × Large White) weaned barrows (7.41 ± 0.06 kg, 28 d old) were assigned randomly to 5 treatments: 1) non-challenged positive control (PC), 2) ETEC F4 (K88⁺)-challenged negative control (NC), 3) NC + kitasamycin at 50 mg/kg + olaquindox at 100 mg/kg + free acidifier (FA) at 5 g/kg, 4) NC + kitasamycin at 50 mg/kg + olaquindox at 100 mg/kg + P(OA + EO) at 1 g/kg (P1), 5) NC + kitasamycin at 50 mg/kg + olaquindox at 100 mg/kg + P(OA + EO) at 2 g/kg (P2). Each dietary treatment had 6 replicates of one piglet each and the study lasted for 3 wk. On d 7, pigs in NC, FA, P1 and P2 were orally dosed with 10 mL of ETEC F4 (K88⁺) culture (1 × 10⁹ CFU/mL). From d 7 to 14 after the ETEC F4 (K88⁺) challenge, P1 increased gain-to-feed ratio (G:F) significantly (P < 0.05) compared with NC and FA groups. From d 14 to 21, P2 increased the average daily gain of pigs (P < 0.05) compared with NC and FA groups. Compared with NC, P2 reduced tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-10 concentrations (P < 0.05) in sera collected at 4 h later after ETEC F4 (K88⁺) challenge. On d 21, P1 increased occludin and zonula occludens-1 protein expression in ileum compared with NC (P < 0.05). After this 3-wk experiment, alpha diversity of gut microbiota was decreased by P2 compared with PC, and P1 increased the relative abundance of Lactobacillus in ileum, cecum and colon (P < 0.05). In conclusion, dietary P(OA + EO) additive at 2 g/kg combined with antibiotics could improve piglet performance and attenuate inflammation, and P(OA + EO) additive at 1 g/kg combined with antibiotics improved intestinal barrier and increased beneficial microbiota composition after an F4 (K88⁺) challenge.     

Specific DNA fragments coding for ST1 and LT1 toxins, and K88 (F4) adhesin in enterotoxigenic Escherichia coli

Ten porcine strains of enterotoxigenic Escherichia coli possessing the K88 (F4) adhesion fimbriae, were selected for study of enterotoxin- and fimbriae-encoding plasmids. Plasmid DNA, separated according to size by gel electrophoresis was transferred to nylon membranes by Southern blotting, and hybridized with enzyme-labelled oligonucleotide probes for ST1 and LT1 enterotoxins, and a 32P-labelled probe for the F4 fimbriae. Plasmids possessing the enterotoxin genes ranged from 50 MDa to 78 MDa in size. The ST1 genes were located on a common 8-MDa EcoR1 restriction endonuclease fragment, while the LT1 genes were located on a 4.5-MDa EcoR1 fragment from the different plasmids. Plasmids with the F4 genes ranged from 50 MDa to 118 MDa in size, but the F4 encoding genes were located on a common 3-MDa HindIII restriction endonuclease fragment. ST1 and LT1 genes were found on the same plasmid in only one strain, LT1 and F4 genes on the same plasmids in 5 strains, while no plasmid contained genes for both ST1 and F4.     

Susceptibility of Chinese Meishan and European large white pigs to enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, K99, or F41

Conventionally raised Chinese Meishan and European Large White pigs were intragastrically challenge exposed with 2.1 x 10(10) enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, F41, or F41 plus K99. In response to challenge exposure with the K88-positive (K88+) organisms, 96% of Large White pigs died within 48 hours, whereas none of the Meishan pigs died. Both breeds of pigs had similar susceptibility to strains bearing 987P or F41. Lastly, Meishan pigs were found to be more susceptible than Large White pigs to a strain expressing K99 and F41. In pigs with diarrhea, challenge-exposure strains intensively colonized the jejunum (10(8) to 10(10) bacteria/g of tissue) and, to less extent, the duodenum (except K88+ strain, which comprised 10(8)/g). In most cases, jejunal concentrations of the challenge-exposure strains were substantially lower in pigs that did not have diarrhea. Half the resistant Meishan pigs eliminated the K88+ strain from the intestines. Colostral antibody titer that agglutinated challenge-exposure strains did not differ between Meishan and Large White gilts. Results indicate that resistance of pigs to the K88+ strain did not extend to enterotoxigenic strains bearing other well-known factors. They indicate, in addition, that genetic resistance to K88+ strains described in pigs in Europe may exist in pigs in China.     

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.     

Early supplementation with Lactobacillus plantarum in liquid diet modulates intestinal innate immunity through toll-like receptor 4-mediated mitogen-activated protein kinase signaling pathways in young piglets challenged with Escherichia coli K88

This study was conducted to investigate the effects of early supplementation during 4 to 18 d of age with Lactobacillus plantarum (LP) in liquid diets on intestinal innate immune response in young piglets infected with enterotoxigenic Escherichia coli (ETEC) K88. Seventy-two barrow piglets at 4 d old were assigned to basal or LP-supplemented liquid diet (5 × 10¹⁰ CFU·kg⁻¹). On day 15, piglets from each group were orally challenged with either ETEC K88 (1 × 10⁸ CFU·kg⁻¹) or the same amount of phosphate-buffered saline. The intestinal mucosa, mesenteric lymph node (MLN), and spleen samples were collected on day 18. Here, we found that LP pretreatment significantly decreased the mRNA relative expression of inflammatory cytokines (interleukin [IL]-1β, IL-8, and tumor necrosis factor-α), porcine β-defensin 2 (pBD-2), and mucins (MUC1 and MUC4) in the jejunal mucosa in piglets challenged with ETEC K88 (P < 0.05). Moreover, LP significantly decreased the ileal mucosa mRNA relative expression of IL-8 and MUC4 in young piglets challenged with ETEC K88 (P < 0.05). Furthermore, the piglets of the LP + ETEC K88 group had lower protein levels of IL-8, secretory immunoglobulin A, pBD-2, and MUC4 in the jejunal mucosa than those challenged with ETEC K88 (P < 0.05). Besides, LP supplementation reduced the percentage of gamma/delta T cells receptor (γδTCR) and CD172a⁺ (SWC3⁺) cells in MLN and the percentage of γδTCR cells in the spleen of young piglets after the ETEC K88 challenge. Supplementation with LP in liquid diets prevented the upregulated protein abundance of toll-like receptor (TLR) 4, phosphorylation-p38, and phosphorylation-extracellular signal-regulated protein kinases in the jejunal mucosa induced by ETEC K88 (P < 0.05). In conclusion, LP supplementation in liquid diet possesses anti-inflammatory activity and modulates the intestinal innate immunity during the early life of young piglets challenged with ETEC K88, which might be attributed to the suppression of TLR4-mediated mitogen-activated protein kinase signaling pathways. Early supplementation with LP in liquid diets regulates the innate immune response, representing a promising immunoregulation strategy for maintaining intestinal health in weaned piglets.