A comparison of the ecology of Escherichia coli in the intestine of healthy unweaned pigs and pigs after weaning

A total of 51 clinically healthy pigs (14 unweaned and 37 weaned) from five litters, and aged 21 to 35 d, were studied. Escherichia coli isolates from the duodenum, jejunum, ileum, caecum and colon were differentiated on the basis of O-serogroup, biotype and resistance pattern. The complexity of the flora was influenced considerably by the presence or absence of the enterotoxigenic serotype 0149: K91, K88a, o (Abbotstown strain). When it was absent the E. coli flora of both weaned and unweaned pigs was complex with up to 25 strains being identified. The majority of these E. coli strains identified in each pig were isolated from only one of the five intestinal sites sampled. On the other hand, when the enterotoxigenic strain was present (14 pigs) it tended to dominate the E. coli flora at all levels of the intestine and this dominance was reflected in a corresponding fall in the total number of E. coli strains isolated per pig.     

Pilus antigen 987P produced by strains of Escherichia coli serotypes O141:K--, H- and O8:K85:H--

Escherichia coli strains isolated from the alimentary tract of 68 weaned and 44 unweaned pigs with diarrhoea in various parts of Hungary, were tested for the presence of pilus antigens K88, K99 and 987P. K88 was detected in 30% of the strains from newborn pigs and in 12% of the strains isolated from weaned pigs. One strain carried K99. Based on agglutination test and immunoelectron microscopic studies with specific absorbed antisera, five non-haemolytic E. coli strains isolated from newborn pigs were found to produce so-called 987P pili. Three of these strains were designated serologically as O8:K85:H--,987P+ and two as O141: K--:H--,987P+. The Y1 cell assay, the infant mouse assay, and the ligated intestinal loop assay in less than 3-week-old pigs indicated that none of the strains produced heat-labile enterotoxin but all produced a heat-stable enterotoxin detectable in infant mice and in pig loops (STa). All the strains induced diarrhoea in newborn, colostrum deprived pigs and colonized the lower small intestine by adhesion to the villous epithelium. The results have confirmed earlier findings about adhesive virulence attributes caused by 987P pili.     

Vero cell toxins in Escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs

Sixty-eight of 519 strains of Escherichia coli and six of 10 strains of Pseudomonas aeruginosa produced toxins acting on Vero cells (VT+); all of 63 Salmonella, Shigella, Klebsiella, Enterobacter and Proteus strains were VT-. Most of the VT+ E. coli strains were from weaned pigs suffering from oedema disease and/or diarrhoea and belonged to serogroups O141:K85,88, O141:K85, O138:K81, and O139:K82; six VT+ E. coli strains were from diarrhoeic human babies, four of serogroup O26 and two of serogroup O128. The VT genes in two of the O26 strains and in the O128 strains were located in the genome of the phages with which they were lysogenized. One O141:K85,88 pig E. coli strain transferred its VT genes, probably by conjugation, to E. coli K12. The VTs of the human E. coli strains, the pig E. coli strains and the P. aeruginosa strains were antigenically different from each other; unlike the others, the P. aeruginosa VT was heat-resistant. Cell-free preparations of cultures of E. coli K12 to which the VT genes of the four human E. coli strains had been transferred caused fluid accumulation in ligated segments of rabbit intestine. Inoculated intravenously, they were lethal for mice and rabbits; similar preparations of E. coli K12 to which the VT genes of the pig E. coli strain had been transferred produced a disease in pigs that clinically and pathologically resembled oedema disease.     

Response of nursery pigs to a synbiotic preparation of starch and an anti-Escherichia coli K88 probiotic

Postweaning diarrhea in pigs is frequently caused by enterotoxigenic Escherichia coli K88 (ETEC). The aim of this study was to test the efficacy of E. coli probiotics (PRO) in young pigs challenged with E. coli K88. We also tested the synbiotic interaction with raw potato starch (RPS), which can be used as a prebiotic. Forty 17-day-old weaned piglets were randomly assigned to four treatments: treatment 1, positive-control diet (C), no probiotics or RPS but containing in-feed antibiotics; treatment 2, probiotic (PRO), no feed antibiotics plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7; treatment 3, 14% RPS, no antibiotics (RPS); treatment 4, 14% RPS plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7, no antibiotics (PRO-RPS). The pigs were challenged with pathogenic E. coli K88 strains on day 7 of the experiment (24-day-old pigs) and euthanized on day 10 of the experiment (35-day-old pigs). Probiotic and pathogenic E. coli strains were enumerated by selective enrichment on antibiotics, and microbial community analysis was conducted using terminal restriction length polymorphism analysis (T-RFLP) of 16S rRNA genes. The combination of raw potato starch and the probiotic had a beneficial effect on piglet growth performance and resulted in a reduction of diarrhea and increased microbial diversity in the gut. We conclude that the use of E. coli probiotic strains against E. coli K88 in the presence of raw potato starch is effective in reducing the negative effects of ETEC in a piglet challenge model.     

The effect of Lactobacillus spp. on the attachment of enterotoxigenic Escherichia coli to isolated porcine enterocytes

A total of 43 strains of lactobacilli were isolated from the gastrointestinal tract of piglets at the time of weaning. Isolates, grown on solid media, were allocated to strongly adherent or non/weakly adherent groups on the basis of numbers attaching to isolated porcine enterocytes. Strains of Lactobacillus fermentum were disproportionally represented amongst the strongly-adherent strains and Lact.acidophilus and Lact. salivarius amongst the non/weakly-adherent group. Lactobacilli showed significantly better attachment ability when grown on agar than when grown in broth culture. Strongly adherent strains were not found to effect the attachment of enterotoxigenic Escherichia coli to porcine enterocytes, tested under the conditions of exclusion (lactobacilli added to the enterocytes before E. coli ), competition (lactobacilli and E. coli added simultaneously) and displacement ( E. coli added before lactobacilli). Tests were made with [¹⁴C]-labelled E. coli. Suspensions of bacteria and enterocytes were passed through a filter selected to retain enterocytes but pass free bacterial cells. Counts (dpm) obtained from filters after solubilization were taken as a measure of E. coli attachment. Some strains of lactobacilli coaggregated with enterotoxigenic E. coli with K88 fimbriae, but not with a K88-negative mutant strain. These were excluded from the competitive exclusion experiments. In the apparent absence of a direct effect on the association of E. coli with host tissue, removal of potential gut pathogens by aggregation could contribute to the probiotic properties ascribed to lactic acid bacteria.     

A probable new adhesive factor (F42) produced by enterotoxigenic Escherichia coli isolated from pigs

Three enterotoxigenic Escherichia coli (ETEC) strains (coded 62, 104, and 567/7) isolated from piglets with neonatal diarrhea produced only a thermostable enterotoxin. Although these strains showed mannose-resistant microhemagglutination (MRMH), the responsible factor was serologically different from the known hemagglutinating colonization factors from porcine strains (K88, K99, and F41). Bacterial cells from these strains adhered to HeLa cells and pig brush borders. Electron microscope studies revealed the presence of fimbria-like structures on bacterial cells grown at 37 C but not on cells grown at 18 C. The antiserum prepared from partially purified fimbrial antigen (provisionally called F42) inhibited chicken erythrocyte MRMH caused by these strains as well as adherence of strain 567/7 to HeLa cells and to pig brush borders. These data taken together suggest the existence of a new hemagglutinating adhesin that is different from those so far described for porcine ETEC.     

大肠杆菌O138 O-抗原基因簇的破译及Gne的生物信息学鉴定

利用鸟枪法对大肠杆菌E .coliO138O 抗原基因簇进行测序 ,序列全长 14 139bp ,用生物信息学的方法进行序列分析 ,共发现 11个基因 ,分别为鼠李糖合成酶基因 (rmlB ,rmlD ,rmlA ,rmlC)、UDP GalNAcA合成酶基因 (gne ,gna)、糖基转移酶基因 (3个 )、O 抗原转运酶基因 (wzx)和O 抗原聚合酶基因 (wzy)。发现一种稀有单糖UDP Gal NAcA的合成途径 ,对合成该糖的第一种酶Gne进行了生物信息学鉴定 ,另外用PCR方法筛选出了针对大肠杆菌O138的特异基因     

Antimicrobial susceptibilities of Shiga toxin-producing Escherichia coli isolates from pigs with edema disease in Japan

Fifty-seven Shiga toxin-producing Escherichia coli (STEC) strains isolated from pigs with edema disease (ED) from 1997 to 2001 in Japan were examined for antimicrobial susceptibilities. The susceptibilities were compared with those of E. coli ATCC 23546 isolated from pig with ED in the 1950's. Consequently, the isolated STECs showed high susceptibility to peptides and bicozamycin in a way similar to the reference strain. On the other hand, the STECs showed low susceptibility to beta-lactams, tetracyclines, novobiocin, fosfomycin, trimethoprim, and old quinolones. It became clear that the susceptibilities of the isolated STECs had diminished in regard to antimicrobials.     

Blood groups and serum protein polymorphisms in the Pitman-Moore and Ohmini strains of miniature pigs

The variations of blood groups and biochemical polymorphisms in the populations of the two miniature pig strains reared in Japan were investigated in this report. The variations in eight blood group systems were investigated by using serological techniques, and five serum protein systems by using starch gel electrophoresis. The Pitman-Moore strain showed no variations in K, O, Tf, Cp and Am systems, but showed plymorphisms in A, E, F, G, H, L, Pa and Hpx systems. The Ohmini strain showed no or little variations in E, F, G, Tf, Pa and Cp systems, but showed polymorphisms in A, H, K, L, O, Hpx and Am systems. From the results of investigation of genetic similarities among nine pig populations including various breeds, it was made clear that the two miniature pig strains were very different genetically, and the Pitman-Moore strain was genetically closer to European large pig breeds than to East Asian native pigs, while the Ohmini strain was close to Thai and Philippine natives.     

Cholesterol 7 alpha-hydroxylase activity and bile acid synthesis in hepatocytes of unweaned and weaned pigs in monolayer culture

Activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7 alpha-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7 alpha-hydroxylase activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [14C]cholesterol, among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7 alpha-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7 alpha-hydroxylase activities and bile acid production rates.     

Application of polymerase chain reaction for determination of toxins in Escherichia coli strains isolated from pigs with diarrhea.

The PCR method was used for the determination of LTI, STI, STII enterotoxins and Stx2e toxin genes in E. coli strains isolated from pigs with diarrhea. It was shown that most of the strains (77.3%) were enterotoxigenic, producing mainly LTI and STII (30.8%) or STI and STII (21.3%) toxins. It was found that 10.6% of isolates possessed the stx2e genes responsible for the elaboration of shiga toxin 2e.     

Drug Resistance in Strains of Escherichia Coli Isolated from the Intestinal Tract of Pigs in Norway

Faecal samples from 95 healthy pigs and samples of jejunal content from 85 piglets suffering from colienterotoxaemia were tested for the presence of drug resistant E. coli strains. Practically all pigs in both groups harboured E. coli strains resistant to one or more of the 6 antibiotics/chemotherapeutic agents tested (Oxytetracycline, streptomycin, sulphaisodimidin, neomycin, ampicillin, chloramphenicol). Almost 100% of healthy and approx. 90% of diseased pigs harboured strains resistant to Oxytetracycline, streptomycin and sulphaisodimidin. Pigs with strains resistant to neomycin, ampicillin and chloramphenicol were less frequently found. The predominant coliform flora consisted of E. coli strains” resistant to Oxytetracycline, streptomycin and sulphaisodimidin in 71% to 81% of diseased pigs and in 47% to 69% of the healthy pigs. In diseased pigs ¾ of the animals had a coliform flora dominated by neomycinresistant E. coli strains. Of the 721 resistant E. coli strains isolated from healthy pigs, 11% were single resistant while the corresponding figure for the 518 resistant strains isolated from diseased pigs was 6%. Thus 89% and 94% of strains showed simultaneous resistance to 2 or more antibiotics. E. coli strains resistant to 3 or more drugs were found in approx. 60% and 70% of the isolates from healthy and diseased animals, respectively. Oxytetracycline/streptomycin/sulphaisodimidin resistance was most commonly found, approx. 22% and 38% of the strains from healthy and diseased pigs, respectively, showing this resistance pattern. Transmission of drug resistance which was examined in E. coli strains originating from the diseased pigs was demonstrated in approx. 76% of the isolates. The incidence of drug resistance transfer in single, double, triple and quadruple resistant strains was 11%, 68%, 97% and 98%, respectively.     

PCR assay for detection and differentiation of K88ab1, K88ab2, K88ac, and K88ad fimbrial adhesins inE. coli strains isolated from diarrheic piglets

Primers were designed and prepared and conditions were determined for PCR detection and differentiation of enterotoxigenic E. coli bacterial strains isolated from diarrheic pigs. Primers K88/1 and K88/2 are 25 bp oligomers that correspond to a region of genes encoding one of serological variants of the K88 antigen (K88ab(1), K88ab(2), K88ac or K88ad). A positive result of PCR is an amplificate of 792 bp in size for K88ab and K88ad variant or 786 bp for K88ac variant. The individual serological variants of genes of the K88 antigen could be differentiated by cutting the obtained PCR amplificates by restriction endonucleases. The PCR analysis of 674 E. coli strains isolated from diarrheic pigs showed that 184 strains were K88 positive. By using restriction endonucleases the K88-positive strains were in 4 cases classified as K88ab variant, 180 as K88ac variant and none contained gene for the K88ad variant. Ninety-five % coincidence with serological examination using K88ab, K88ac and K88ad specific antibodies was shown.     

Enterotoxigenicity and frequency of Campylobacter jejuni, C. coli and C. laridis in human and animal stool isolates from different countries

Campylobacter jejuni and C. coli strains were collected during three different years from adult patients with enterocolitis in Sweden (n = 372) from 49 patients in Kuwait, and Campylobacter strains from hens from Mexico, Pakistan and Sweden (n = 107) and Swedish pigs (n = 47). C. jejuni was the predominant species in human and hen isolates, and C. coli in pigs C. coli was significantly more common in human isolates from Sweden, and more common in hen isolates from Pakistan, than in hens from Sweden and Mexico. C. laridis was only isolated from pigs (17%) and was in no case enterotoxigenic. Both in human and hen isolates, C. jejuni strains were more enterotoxigenic than C. coli strains. C. jejuni strains from Swedish hens were less enterotoxigenic than those from Pakistan and Mexico (P less than 0.001), and strains from pigs were less enterotoxigenic than those from hens (P less than 0.001). We conclude that C. jejuni are more often enterotoxigenic and possibly more virulent than c. coli and C. laridis. The relative frequency of C. jejuni and C. coli in humans and animals differs from one country to another.     

Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13

Diarrhoea in newborn and weaned pigs caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae leads to considerable losses in pig production. In this study, we refined the mapping of the receptor locus for ETEC F4ab/F4ac adhesion ( F4bcR ) by joint analysis of Nordic and Swiss data. A total of 236 pigs from a Nordic experimental herd, 331 pigs from a Swiss experimental herd and 143 pigs from the Swiss performing station were used for linkage analysis. Genotyping data of six known microsatellite markers, two newly developed markers ( MUC4gt and HSA125gt ) and an intronic SNP in MUC4 ( MUC4-8227 ) were used to create the linkage map. The region for F4bcR was refined to the interval SW207 – S0075 on pig chromosome 13. The most probable position of F4bcR was in the SW207 – MUC4 region. The order of six markers was supported by physical mapping on the BAC fingerprint contig from the Wellcome Trust Sanger Institute. Thus, the region for F4bcR could be reduced from 26 to 14 Mb.     

Echinococcus granulosus from Mexican pigs is the same strain as that in Polish pigs

Samples of Echinococcus granulosus from seven pigs from Mexico were compared with isolates of the parasite from pigs in Poland and representative strains and species of Echinococcus. Isolates from pigs in Mexico were found to be genetically identical to E. granulosus from Polish pigs and distinct from other major genotypes by sequencing part of the mitochondrial cytochrome c oxidase I (COI) mtDNA locus, restriction fragment length polymorphism (RFLP) of the polymerase chain reaction (PCR) amplified rDNA internal transcribed spacer (ITS) 1 using five different enzymes, and random amplified polymorphic DNA (RAPD) analysis. These results were complemented by data on hook morphology and together strengthen the view that Echinococcus maintained in a cycle involving pigs and dogs is a distinct strain that is conserved genetically in different geographical areas. The present study supports the close relationship of the cervid, camel and pig strains and raises the question of their taxonomic status.     

Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets

The protective effects of egg-yolk antibodies obtained from hens immunized with fimbrial antigens from a local strain (Escherichia coli K88+ MB, Manitoba, Canada) of K88+ piliated enterotoxigenic E. coli (ETEC) were evaluated in 3- and 21-day-old piglets in which ETEC diarrhea was induced and also in early-weaned piglets in a commercial farm. The results demonstrated that the E. coli K88+ MB-induced diarrhea in 3-day-old piglets was cured 24 h after treating with egg-yolk antibodies while those treated with egg-yolk powder from conventional hens continued to have diarrhea and 62.5% of them died of severe diarrhea. For 21-day-old weaned piglets, those fed egg-yolk antibodies had transient diarrhea, positive body weight gains and 100% survival during the period of the experiment, whereas control piglets that were treated with placebo had severe diarrhea and dehydration and some died within 48 h after infection. In the field trial, the incidence and severity of diarrhea of 14-18-day-old weaned piglets fed egg-yolk antibodies were much lower than in those fed a commercial diet containing an antibiotic. These results indicate that the neonatal and early-weaned piglets that received the egg-yolk antibodies were protected against ETEC infection.     

The gram-positive tonsillar and nasal flora of piglets before and after weaning

AIMS: To investigate gram-positive nasal and tonsillar microbial flora of piglets before and after weaning. METHODS AND RESULTS: The nasal and tonsillar gram-positive bacterial flora of 20 non-weaned piglets (2 weeks of age) and 20 weaned piglets (6 weeks of age), obtained from four different piggeries, was quantified by culture and identified by tDNA-PCR. The most widely occurring species from nasal conchae before as well as after weaning in the different piglets investigated were Streptococcus suis and Rothia nasimurium. After weaning a wide variety of Lactobacillus species appeared but in low numbers. In the tonsils, Strep. suis, Strep. dysgalactiae, S. hyicus, S. aureus, Arcanobacterium pyogenes and Actinomyces hyovaginalis were the species isolated from the largest number of pigs before and after weaning. S. aureus and most lactobacilli became more prevalent after weaning. Bacteria not known to be associated with pigs found in the present study included R. nasimurium, Strep. gallolyticus, Pediococcus pentosaceus and some Lactobacillus species. CONCLUSIONS: Over 30 different gram-positive bacterial species may occur in nasal conchae and tonsils of unweaned piglets at 2 weeks of age and of 6-week-old weaned piglets. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that weaning is associated with changes in prevalence of only a small minority of the highly diversified bacterial flora of the nares and tonsils of pigs.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

Clonal relationships among Escherichia coli serogroup 06 isolates from human and animal infections

The clonal relationship of thirty E. coli strains of 0 antigen serotype 06 isolated from human, dog, pig or cow infections were investigated. Two main clones with serotypes 06 : H1 or 06 : H31, H- were identified. Isolates from humans, dogs, pigs and cows were found in both clones, indicating that animals are a possible source for human extraintestinal Escherichia coli strains. Two human ETEC (06 : H16) and two pig isolates (06 : H10) were not related to the 06 : H1 or 06 : H31, H- E. coli clones.