Escherichia coli K88 receptor expression in intestine of disease-susceptible weaned pigs.

A challenge trial was carried out in which Escherichia coli O157 K88ac was administered to a litter of weaned pigs and the development of the disease monitored over a five-day experimental period. The eight animals in the trial were assigned to two groups depending on whether they exhibited disease symptoms. Six pigs developed diarrhoea and two appeared unaffected; these were designated as the test (or K88-susceptible) group and the control (or K88-resistant) group, respectively. The animals were euthanised and the intestine was removed and sections processed for brush border membrane vesicle preparation. Microscopic and biochemical assays were undertaken on tissue samples from each animal and a strong correlation was observed between the expression of a glycoprotein receptor complex associated with the brush border membrane and the development of disease symptoms. Further investigation revealed the presence of an analogous glycoprotein complex in the K88-resistant group which did not bind the K88-fimbriae antigen. These results suggest that genetic differences in the glycosyl moieties of the receptor complex provide the basis for disease susceptibility to K88-positive E. coli.     

Levamisole mucosal adjuvant activity for a live attenuated Escherichia coli oral vaccine in weaned pigs

The present study tested the hypothesis that levamisole exerts its immunopotentiating activity in weaned pigs vaccinated against colibacillosis by priming the lymphocytes and macrophages in the mesenteric lymph node (MLN). Ten weaned piglets were used and allocated into two equal groups. The experimental group was intramuscularly primed with levamisole at an immunostimulatory dose of 2.5 mg/kg given daily, in three consecutive days, and controls received saline according to the same schedule. Both groups were orally vaccinated with the vaccinal Escherichia coli strain on day 0 and challenged with the virulent E. coli strain 7 days later. All pigs were killed on postchallenge day 6. Upon virulent challenge the health status of the two groups was evaluated by clinical observations, and expression of CD25, SWC7 and SWC9 activation antigens by MLN and spleen T and B cells and macrophages, respectively, was tested using flow cytometry. Priming by levamisole significantly contributed to the effectiveness of a live attenuated oral vaccine against porcine postweaning colibacillosis, as evidenced by a good health status of primed vaccinated vs. un-primed vaccinated pigs. The CD3+, CD25+ and SWC9+ MLN but not spleen T cells and macrophages increased in experimental vs. control pigs, implying that levamisole exerts its potentiating activity in the MLN by augmenting both recruitment and activation of cells that participate in cell-mediated immunity.     

Spray-dried plasma improves growth performance and reduces inflammatory status of weaned pigs challenged with enterotoxigenic Escherichia coli K88

We investigated whether spray-dried plasma (SDP) improved growth and health of piglets challenged with enterotoxigenic Escherichia coli K88 (ETEC). Forty-eight pigs weaned at 21 d (BW = 4.88 +/- 0.43 kg) received one of four diets containing 6% SDP or fish proteins (as-fed basis) either nonmedicated (SDP-NM and FP-NM diets) or medicated with 0 or 250 mg/kg of colistine + 500 mg/kg of amoxycycline (SDP-M and FP-M diets), for 15 d. On d 4, pigs were orally challenged with ETEC. On d 15, eight pigs per dietary group were killed, blood and saliva were collected for analysis of K88 fimbriae-specific immunoglobulin (Ig)-A, and jejunum was removed for villi preparation, histological analysis, and cytokine expression. The presence or absence of K88 receptors (K88+ and K88- pigs respectively) was determined by villous adhesion assay. Effects of protein source on ADG (P = 0.04) and ADFI (P < 0.01), as well of medication on ADFI (P < 0.02), of all pigs were observed. In sacrified pigs, there was an effect of protein source on ADG (P = 0.03) and ADFI (P < 0.001), as well an interaction between medication and presence of K88 receptor (P = 0.02) for feed:gain ratio. Plasma K88 specific IgA were low in all K88 pigs and higher in K88+ pigs fed FP-NM compared with all the other groups (P < 0.05), except SDP-M. An interaction was found among protein source, medication, and presence of K88 receptors (P = 0.04). Saliva IgA concentrations were high in all pigs fed FP-NM and low in all other pigs. Jejunum of pigs fed FP-NM showed some ulcerations, edema, and mild inflammatory cell infiltration (ICI). In pigs fed FP-M, edema was reduced. Conversely, only a mild ICI was observed in pigs fed SDP-NM and SDP-M. Crypt depth was increased in K88+ pigs fed SDP-NM and an interaction between protein source and presence of K88 receptors was observed (P < 0.05). Expressions of tumor necrosis factor-alpha and interleukin (IL)-8 were lower in pigs fed SDP-NM and SDP-M than in those fed FP-NM and FP-M, either K88- or K88+ (P < 0.01). In pigs fed FP diets, expression of IL-8 tended to increase (P = 0.08) in K88+ compared with K88- subjects. Expression of interferon-gamma increased in K88 and K88+ pigs fed FP-M as compared with other pigs (P < 0.01). These results indicate that feeding with SDP improved growth performance and protected against E. coli-induced inflammatory status, and suggest that use of SDP-NM can be considered a valid antibiotic alternative.     

检测K88~+肠毒素性大肠杆菌PCR方法的建立

以K8 8菌毛结构基因保守序列为靶序列 ,设计合成了一对可扩增长 2 0 1bp的目的片段的引物 ,成功地建立了检测肠毒素性大肠杆菌(ETEC)K88菌毛基因的PCR方法。进行了PCR方法的特异性试验和敏感性试验。对K99+ ,F41 + ,987p+ 参考菌株和鼠伤寒沙门氏杆菌 ,链球菌 ,金黄色葡萄球菌和猪肺疫巴氏杆菌的检测结果均为阴性 ;该检测方法的敏感度可达 1 0个细菌。用此方法对 1 0株腹泻仔猪粪便分离物进行检测 ,结果有 2株阳性 ;与血清学检测的结果一致。结果表明此方法特异性和敏感性都很高 ,可用于临床K88+ 肠毒素性大肠杆菌病的快速诊断和流行病学调查     

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.     

Prevalence of K88, K99, and 987P pili of Escherichia coli in neonatal pigs with enteric colibacillosis

One hundred nineteen live neonatal pigs with diarrhea less than or equal to 2 weeks old were euthanatized, and frozen sections of their ilea were submitted to an indirect fluorescent antibody technique to identify K88, K99, and 987P pili (also referred to as F-4, F-5, and F-6 pili, respectively) in Escherichia coli. Ten-centimeter ileal sections were used to determine numbers of lactose-fermenting bacteria. Of 52 pigs in which E coli pili were found, 14 had K88 (27%), 23 had K99 (44%), 13 had 987P (25%), and 2 had K88 and K99 simultaneously (4%). Numbers of lactose-fermenting bacteria were significantly (P less than or equal to 0.05) higher in pigs with piliated E coli than in pigs without piliated E coli. Results of this study indicated that piliated E coli are a major cause of enteric disease in neonatal swine in Michigan, and that in pigs less than or equal to 2 weeks of age, K99 was the most frequently encountered pilus antigen.     

Evaluation of a combined rotavirus and enterotoxigenic Escherichia coli vaccine in cattle

A vaccine of rotavirus and K99 antigen from enterotoxigenic Escherichia coli was emulsified in oil adjuvant and administered intramuscularly to pregnant cows. Calves born to and reared on vaccinated dams were protected against experimental rotavirus infection at five days old when compared with calves from unvaccinated control cows. Field trials of the vaccine were carried out in 40 commercial herds, in which half the cows in each herd were selected at random for vaccination and half were left unvaccinated. In 31 herds (2641 cows) there was no significant diarrhoea problem (less than 10 per cent morbidity); these herds were excluded from further analysis. The nine remaining herds did experience a calf diarrhoea problem of greater than 10 per cent morbidity, but on four farms the disease was associated with cryptosporidiosis and on a fifth no enteropathogens were detected; these five farms (461 cows) were also excluded from further analysis. Of the remaining four herds, two beef suckler herds (105 cows) had concurrent rotavirus and cryptosporidial infections, and vaccination was associated with a decreased excretion of rotavirus but not with a decreased incidence of diarrhoea. In the other two dairy herds (68 cows) with prevaccination rotavirus problems, there was a significantly decreased incidence of diarrhoea in calves born to vaccinated cows. No natural field challenge of enterotoxigenic E coli was encountered on any of the trial farms.     

产肠毒素性大肠杆菌K88与K99菌毛蛋白的串联表达

根据GenBank中发表的E.coli K88、K99基因序列,分别设计合成1对引物.利用PCR技术,以大肠杆菌C83907和C83644的质粒为模板分别扩增不含信号肽的K88及K99基因.通过分离、纯化、限制性核酸内切酶酶切,连接和转化,构建了含K88-K99串联表达载体的重组菌株BL21(DE3)(pETK88CK99).结果显示,经酶切,PCR鉴定和DNA序列分析,证实了构建的重组质粒pETK88CK99中含有K88K99融合基因,且基因序列和阅读框架均正确.经过SDS-PAGE分析,串联表达蛋白含量占菌体蛋白的40%左右,经Western blotting检测,该串联表达蛋白能被大肠杆菌K88、K99标准血清识别.结果表明,构建的重组菌株可以作为预防新生仔猪大肠杆菌性腹泻基因工程疫苗的候选菌株.     

Escherichia coli diarrhoea in pigs with or without the K88 receptor.

There was a high incidence of neonatal scours in 38 litters of pigs born at Compton in a four month period during 1978. The most important cause of the disease was an enteropathogenic Escherichia coli strain which possessed the K88 antigen. The Compton herd has been bred to produce pigs of three genotypes with respect to the presence or absence of the intestinal receptor for the K88 antigen. These are homozygous dominants (SS) and heterozygotes (Ss) susceptible to infection by virulent K88-positive E coli, and homozygous recessives (ss) resistant to the disease. The highest incidence of diarrhoea was in the susceptible progeny of resistant dams and susceptible sires. There was no K88 associated diarrhoea in resistant progeny or in susceptible progeny of susceptible dams.     

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.     

Experimental model of enterotoxigenic Escherichia coli infection in pigs: potential for an early recognition of colibacillosis by monitoring of behavior.

The hypothesis that altered behavior is a sign for an early recognition of disease was tested. The experiment was conducted to evaluate the behavioral patterns of pigs in a model of postweaning colibacillosis. Twenty-five weaned pigs (from a herd that was previously found to be highly susceptible to F4⁺ Escherichia coli strains) were randomly assigned into 5 groups, kept in isolated pens under the controlled ambiental conditions. One day after weaning, the pigs from three groups were intragastrically inoculated (via orogastric tube) with either F4ac⁺ (1466 or 2407) or F4⁻ (1467) nonenterotoxigenic E. coli (non-ETEC) strains, respectively. The pigs from the fourth group were inoculated with F4ac⁺ ETEC strain M1823 and the remaining 5 pigs that received broth containing 1.2% sodium bicarbonate were kept as noninoculated controls. The pigs were examined daily and the frequency and duration of their behavioral patterns, such as eating, drinking, lying, standing, urinating, defecating, rooting and playing were monitored for 300 h during a period of 10 days. In this model, three conditions were also observed in F4-susceptible pigs: (1) acute fatal diarrheal disease; (2) moderate diarrhea and weight loss and (3) no diarrhea and weight loss. The incidence (both frequency and duration) of defecating was significantly higher (P<0.05) in pigs inoculated with F4ac⁺ ETEC strain M1823 as compared to that of noninoculated (control) pigs. Pigs inoculated with F4ac⁺ non-ETEC strain 1466 had a significantly lower frequency of eating (P<0.05) and frequency/duration of drinking (P<0.05) than did the controls. The 1466-inoculated pigs, had an increased diarrhea score, but frequency/duration of defecating was not significantly different. Pigs inoculated with F4ac⁺ non-ETEC strain 2407 spent more time in lying (P<0.05) than did noninoculated pigs. Conversely, the pigs that received F4⁻ non-ETEC strain 1467 laid shorter (P<0.05) and ate/drank less frequently (P<0.05) than the controls. It was concluded that the changed occurrence of defecating and eating in pigs that were inoculated with either F4ac⁺ ETEC (M1823) or non-ETEC (1466) strain, respectively, was consistent with the pending clinical disease, i.e. postweaning colibacillosis.     

Colonization of the small intestine of weaned pigs by enterotoxigenic Escherichia coli that lack known colonization factors.

Intestinal colonization of 3-week-old weaned pigs by enterotoxigenic Escherichia coli (ETEC) strains that were originally isolated from weaned pigs with fatal diarrhea and that lacked K88, K99, F41, and 987P adhesins (4P- ETEC) was studied by histologic, immunofluorescent, and electron microscopic techniques. In the first experiment, 16 principal pigs were inoculated orogastrically with ETEC strain 2134 (serogroup O157: H19) or 2171 (serogroup 0141:H4), and eight control pigs were not inoculated. In the second experiment, 24 principals were inoculated with ETEC strain 2134, and 12 controls were inoculated with a nonenterotoxigenic strain of E. coli. Principal and control pigs were necropsied at intervals from 24 to 72 hours after inoculation of principals to provide the tissues used for this report. Results from the two experiments and with both ETEC strains were similar and therefore were combined. Adhesion by 4P- ETEC was demonstrated in ileum but not in cecum or colon in 22/40 principal pigs sampled at 24 to 72 hours after orogastric inoculation. Adherent bacteria were most apparent on the intestinal villi covering Peyer's patches. Only occasional adherent bacteria were detected in ileal sections from a few (4/20) of the control pigs. Adherence by 4P- ETEC was characterized by "patches" of bacteria closely associated with the lateral surfaces and less frequently with the tips and the bases of intact villi. In most cases, the adherent bacteria were separated from epithelial cell microvilli and other bacterial cells by a 50-400-nm space. Filamentous bacterial appendages bridged this space and formed a network among adjacent bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of enterotoxigenic Escherichia coli in piggeries in Victoria by DNA hybridisation using K88, K99, LT, ST1 and ST2 probes

Rectal swabs collected from piglets with diarrhoea from commercial pig farms were examined for the presence of enterotoxigenic Escherichia coli (ETEC) using DNA hybridisation methods. The probes specifically detected genes for the K88 and K99 fimbrial antigens and the heat-labile and heat-stable enterotoxins. DNA hybridisation methods detected more ETEC than could be detected by either enzyme-linked immunosorbent assay (ELISA) or slide agglutination methods, and also offered the opportunity to test for fimbrial antigens and toxins concurrently. The DNA hybridization method was shown to be applicable to ETEC detection in mixed growths cultured directly from rectal swabs to filters. The method eliminates the need for toxin tests using animals and enables very large numbers of samples to be investigated. The use of toxin probes has revealed large numbers of ETEC with uncharacterized fimbrial antigens.     

产肠毒素大肠杆菌K88ab/K88ad菌毛操纵子fae全基因的克隆、表达及生物学活性的初步研究

为研究K88ab/K88ad菌毛对细胞的黏附作用,本研究分别以产肠毒素E.coli (ETEC) K88ab C83901株和K88ad C83903株基因组DNA为模板,采用PCR技术扩增这两种K88菌毛操纵子fae基因(均约7.9 kb).将其分别克隆于表达质粒pBR322中构建pBR-K88ab和pBR-K88ad重组质粒,并将其分别转化至不含任何菌毛的E.coli SE5000株中.该重组菌能够分别与鼠抗K88菌毛阳性血清和抗K88菌毛单克隆抗体(MAb)产生凝集反应;在电镜下观察到重组菌表面大量表达K88菌毛.采用热抽提法提取其体外表达的K88ab和K88ad菌毛,SDS-PAGE电泳检测结果显示,菌毛蛋白的分子量约为26 ku.玻板凝集试验和western blot结果表明:重组表达的K88ab及K88ad菌毛与K88+参考株菌毛均能够被抗K88菌毛阳性血清和MAb识别.以猪小肠上皮细胞系IPEC-J2为模型进行黏附和黏附抑制试验,结果表明表达K88菌毛的重组菌及K88+参考株均能够黏附于IPEC-J2上皮细胞表面;而且阳性血清和MAb能够有效抑制重组菌或K88+参考株对猪小肠上皮细胞系的黏附结合.     

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.     

Effects of an orally administered live Escherichia coli pilus vaccine on duration of lacteal immunity to enterotoxigenic Escherichia coli in swine

Primigravid swine were vaccinated orally with a live enterotoxigenic Escherichia coli (ETEC) strain that produces pilus antigen K99. The titers of K99 antibody in colostrum and milk of vaccinates remained higher than those of nonvaccinated controls through the first lactation after vaccination (4 weeks). Some control swine had low titers of K99 antibody in colostrum or developed low titers of K99 antibody in milk during lactation. Lacteal K99 antibody titers of vaccinates dropped to control levels during the second lactation, 6 months after vaccination. Pigs suckling vaccinates and controls were equally susceptible to challenge exposure to K99+ ETEC during the second lactation. Orally vaccinated swine given a parenteral booster vaccination (with killed K99+ ETEC) during their second gestation had K99 antibody in milk through their second lactation. During the second lactation, these orally vaccinated parenterally revaccinated swine had higher titers of K99 antibody in postcolostral milk than did nonvaccinated controls, controls given only the parenteral booster injection, or controls vaccinated parenterally during both gestations.