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.     

Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro

The role of flagella in the pathogenesis of F4ac⁺ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference {"type":"entrez-nucleotide","attrs":{"text":"C83902","term_id":"2706834","term_text":"C83902"}}C83902 ETEC strain (O8:H19:F4ac⁺ LT⁺ STa⁺ STb⁺), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac⁺ ETEC adhesion in vitro.     

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.     

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.     

Prevalence of fimbial colonization factors F18ab and F18ac in Escherichia coli isolates from weaned piglets with edema and/or diarrhea in China

F18ab and F18ac are important fimbrial colonization factors of verotoxigenic Escherichia coli (VTEC) and/or enterotoxigenic E. coli (ETEC) in weaned piglets with edema disease and/or diarrhea. To further investigate their prevalence and correlation to pathogenic E. coli, a duplex PCR, using three primers derived from the nucleotide sequence of the F18 major fimbrial subunit gene (fedA), and a direct agglutination test, using a monoclonal antibody specific for the antigenic factor 'a' of F18, were performed. Among 60VTEC, 24VTEC/ETEC and 24 ETEC isolates tested from weaned piglets with edema disease and/or diarrhea in different pig farms in the Jiangsu Province of China, 52 isolates (48.15%) were positive in the direct agglutination test and 63 isolates (58.33%) were positive in the duplex PCR. Among 63 PCR-positive isolates, 53 isolates (49.07%) were F18ab-positive and 10 isolates (9.26%) were F18ac-positive. In addition, the F18ab gene was more frequently detected in VTEC (61.67%) or VTEC/ETEC (62.50%) than in ETEC (4.17% only), while the F18ac gene was more frequently detected in VTEC/ETEC (33.33%) than in ETEC (8.33%) or VTEC (0%). Furthermore, F18ab was more frequently associated with Shiga toxin 2e (Stx2e), whereas F18ac was more frequently associated with enterotoxin ST I. These results suggest that the duplex PCR performed in this experiment is a more reliable method for identification of F18+E. coli, and that F18 is a more important virulence factor of VTEC and VTEC/ETEC.     

Genotypic prevalence of F4 variants (ab, ac, and ad) in Escherichia coli isolated from diarrheic piglets in Korea.

A total of 812 Escherichia coli strains isolated from diarrheic piglets were tested for the presence of the F4 (K88) variant (ab, ac and ad) gene by the polymerase chain reaction. Forty four (5.4%) of the 812 E. coli strains carried genes for F4. Among the 44 isolates known to carry genes for F4, 42 (96%) isolates contained genes for F4ac and 2 (4%) isolates contained genes for F4ab. None of the E. coli strains carried genes for F4ad. Our data show that F4ac is the predominant F4 variant associated with diarrhea in piglets in Korea.     

Characterization of polymorphisms of transferrin receptor and their association with susceptibility to ETEC F4ab/ac in pigs

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 (F4ab, F4ac and F4ad) fimbriae is a significant cause of mortality and morbidity in newborn and weaned pigs. The locus controlling susceptibility towards ETEC F4ab/ac has been mapped to SSC13q41, in which TFRC (transferrin receptor) was localized and considered as a positional candidate gene for ETEC F4ab/ac receptor. In this study, we determined susceptibility/resistance to ETEC F4ab/ac in a total of 755 F2 animals from a White Duroc x Erhualian intercross using a microscopic enterocyte adhesion assay. We identified two TFRC polymorphisms (SNPs 591 A>G and 632 A>G) in a single exon after comparative sequencing analysis of 2371-bp amplicons containing the complete coding region of TFRC using RNA of eight full-sib F2 animals with susceptible and resistant phenotypes. The intron sequences flanking the two exon polymorphisms were obtained, revealing an intron polymorphism (SNP 291 C>T). We genotyped the 19 founder animals of the White Duroc x Erhualian intercross for the identified polymorphisms, showing that only the 291 C>T polymorphism is a highly informative marker. We further genotyped all 59 F1 and 755 F2 animals for the 291 C>T polymorphism, and the association of this polymorphism with susceptibility/resistance to ETEC F4ab/ac in these F2 animals was evaluated by the transmission disequilibrium test. The result showed that the 291 C>T polymorphism is not a causal mutation, however, has a significant linkage disequilibrium with the ETEC F4ab/ac, especially F4ac receptor locus.     

Protection against neonatal Escherichia coli diarrhoea in pigs by vaccination of sows with a new vaccine that contains purified enterotoxic E. coli virulence factors F4ac, F4ab, F5 and F6 fimbrial antigens and heat-labile E. coli enterotoxin (LT) toxoid

The efficacy of a new vaccine against neonatal Escherichia coli diarrhoea in piglets containing purified F4ab, F4ac, F5 and F6 fimbriae and detoxified heat-labile toxin (LT) was tested in challenge experiments by the method described by the European Pharmacopoeia (3rd edn, EDQM, Council of Europe, Strasbourg, France). A group of 11 young sows from a herd without E. coli problems was vaccinated 6-8 and 2-4 weeks prior to expected farrowing and another group of nine young sows were non-vaccinated controls. Escherichia coli antibody titres were determined in serum samples taken from the sows before first vaccination and before farrowing and in colostrum samples. The newborn piglets were allowed to suckle colostrum from their mother immediately after birth. The piglets were marked with individually numbered ear tags. Approximately 12 h after birth, 118 piglets from vaccinated sows and 79 piglets from non-vaccinated control sows were challenged by oral instillation of 5 ml of a freshly prepared culture of one of the challenge strains [O8:K87:F4ab (LT+) or O149:K91:F4ac (LT+) or O9:K30:F5 or O9:K103:F6 respectively]. The challenge cultures contained as a mean 6.8x10(9) CFU/ml. After challenge the piglets were observed for 7 days and mortality and morbidity were recorded. Vaccinated sows developed significant levels of antibody titres in colostrum and serum. Control sows stayed at a low/seronegative level. The protective efficacy was excellent because 66.7-87.5% of the piglets from vaccinated sows remained without clinical signs after challenge. Only 0.0-28.0% of the piglets from non-vaccinated sows remained healthy and more than 47.1% of the piglets in this group died after challenge. It is concluded that the new vaccine is very effective in protection of piglets against neonatal E. coli diarrhoea.     

产肠毒素大肠杆菌K88诱发BALB/c鼠肠炎模型的建立及评价

为建立BALB/c小鼠感染产肠毒素大肠杆菌(ETEC)K88肠炎模型,本研究分别灌服小鼠浓度为1.0×107cfu/m L、1.0×108cfu/m L和1.0×109 cfu/m L的菌液,连续灌服3 d,对其发病情况和外周血中各细胞因子水平进行检测。结果显示,灌菌后小鼠食欲下降、体重降低,外周血中IL-1α、TNF-α以及髓过氧化物酶(MPO)含量增加,十二指肠病理组织切片显示炎症发生。灌菌期结束后第5 d,小鼠粪便稀软,体重降到最低,所检测的细胞因子含量达到峰值,与对照组差异极显著(p0.05);十二指肠肠黏膜层与肌层间隙增宽、杯状细胞增多、固有层与黏膜层炎性细胞侵润,炎症变化最为明显。本研究建立了BALB/c小鼠感染ETEC K88肠炎模型,于灌菌期结束后第5 d炎症最为严重。此外,接种不同浓度菌液的小鼠炎性因子数量变化差异不显著(p0.05)。     

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 .     

Effect of heat stable and heat labile Escherichia coli enterotoxins and cholera toxin in combination with theophylline on unidirectional sodium and chloride flux in the small intestine of weanling swine.

The effect of heat stable and heat labile Escherichia coli enterotoxins or cholera toxin in combination with theophylline on net water, sodium and chloride and unidirectional sodium and chloride fluxes was examined in acute isolated loops of jejunum of weanling swine. The effect of heat stable enterotoxin in combination with theophylline was determined in loops located in the proximal jejunum, while combinations of theophylline and either heat labile enterotoxin or cholera toxin were studied in the distal jejunum. In each situation the addition of theophylline resulted in an additive rather than a synergistic increment of intestinal secretory activity. This study implies that the intestinal adenyl cyclase system and enterotoxin induced intestinal secretion may not be directly related in the swine small intestine.     

Investigation of the relationship between SLA-1 and SLA-3 gene expression and susceptibility to Escherichia coli F18 in post-weaning pigs

Porcine post-weaning diarrhea and edema disease are principally caused by Escherichia coli strains that produce F18 adhesin. FUT1 genotyping and receptor binding studies divided piglets into E. coli F18-resistant and -sensitive groups, and the roles of SLA-1 and SLA-3 were investigated. SLA-1 and SLA-3 expression was detected in 11 pig tissues, with higher levels of SLA-1 in lung, immune tissues and gastrointestinal tract, and higher levels of SLA-3 also in lung and lymphoid tissues. Both genes were expressed higher in F18-resistant piglets, and their expression was positively correlated in different tissues; a negative correlation was observed in some tissues of F18-sensitive group, particularly in lung and lymphatic samples. Gene ontology and pathway analyses showed that SLA-1 and SLA-3 were involved in 37 biological processes, including nine pathways related to immune functions. These observations help to elucidate the relationship between SLA class I genes and E. coli F18-related porcine gastrointestinal tract diseases.     

Pharmacokinetics of amoxicillin after oral administration in recently weaned piglets with experimentally induced Escherichia coli subtype O149:F4 diarrhea

OBJECTIVE: To measure the effect of Escherichia coli subtype O149:F4-induced diarrhea on the pharmacokinetics of orally administered amoxicillin in affected piglets relative to that of uninfected piglets. ANIMALS: 22 healthy 4-week-old recently weaned Danish crossbred piglets. PROCEDURE: 12 piglets were orally inoculated through gastric intubation with 10(9) CFUs of an E. coli O149:F4 strain and responded by developing diarrhea 12 to 16 hours later. Piglets were dosed with amoxicillin trihydrate solution (20 mg/kg) by gastric intubation. A control group of 10 age-matched piglets without signs of diarrhea was dosed similarly. Blood samples were obtained before amoxicillin administration and at 0.5, 1, 1.5, 2, 3, 6, 12, and 24 hours after amoxicillin administration. The plasma concentration of amoxicillin was analyzed by high-performance liquid chromatography. RESULTS: A significant 39% decrease in the area under the plasma concentration versus time curve of amoxicillin was observed in piglets with diarrhea relative to that of control piglets. The maximum plasma concentration (Cmax) was significantly (52%) lower in piglets with diarrhea, compared with control piglets, while the elimination rate constant, time to reach Cmax, and elimination half-life were unchanged. CONCLUSIONS AND CLINICAL RELEVANCE: Escherichia coli-induced diarrhea may decrease systemic bioavailability of amoxicillin. Escherichia coli bacteria attach to the intestinal epithelial cells. Because it is assumed that the concentration of the antimicrobial at the site of infection reflects the systemic concentration, higher doses of amoxicillin in the treatment of piglets with E. coli O149:F4-induced diarrhea may be appropriate.     

Comparison of production traits between pigs with and without the Escherichia coli F4 receptors in a White Duroc x Erhualian intercross F2 population

To evaluate the influence of enterotoxigenic Escherichia coli (ETEC) F4 receptors on production traits in pigs, ETEC F4ab, F4ac, and F4ad adhesion phenotypes and 27 traits related to growth, carcass, meat quality, and length of the small intestine in a White Duroc x Erhualian intercross population were measured. Performance data revealed that pigs with the F4ab or F4ac receptor (adhesive phenotypes) had greater (P < 0.01) ADG during the fattening period (from 46 to 240 d) and carcass weight and length at 240 d than pigs lacking the receptors (nonadhesive phenotype). Conversely, animals having the F4ad receptor had less (P < 0.01) ADG during the fattening period and carcass weight than those lacking the receptor. In total, 8 adhesion patterns (A to H) for the 3 F4 strains were observed in this experimental population. Pigs with both F4ab and F4ac receptors (phenotype B) had greater (P < 0.01) ADG, carcass weight, and length at 240 d compared with pigs without the F4 receptors. No difference was found (P > 0.05) in traits related to meat quality, fatness, and length of the small intestine between pigs with or without the receptors. On the basis of the antagonistic relationship between susceptibility to F4ab/ac and production traits, we speculate that the prevalence of the ETEC F4ab/ac adhesive phenotype in pig populations is attributable to balanced natural and artificial selection.