Haemagglutination patterns of the different variants of Escherichia coli K88 antigen with porcine, bovine, guinea pig, chicken, ovine and equine erythrocytes

Determination of the porcine adhesive phenotype was not achieved by haemagglutination (HA) of porcine erythrocytes, which in all cases were agglutinated by K88ab and K88ad, independent of the adhesive phenotype as determined by the brush border adhesion test. K88ac always gave negative HA results with porcine red cells. However, HA appeared to offer a method of differentiating between the K88 variants without monospecific antisera. K88ab agglutinated porcine, guinea pig and chicken erythrocytes; K88ac agglutinated only guinea pig red cells and K88ad produced haemagglutination with porcine and guinea pig erythrocytes.     

Attachment of E. coli-bearing K88 antigen to equine brush-border membranes

Equine small intestinal brush-border membranes, from 40 adult horses were tested in vitro for the presence of receptors for the Escherichia coli adhesive antigens K88ab, K88ac and K99. Only K88-positive strains of E. coli adhered strongly to horse brush-border membranes. In contrast, a K88-negative mutant strain J2, 2 K99-positive strains and 3 E. coli strains isolated from foals failed to adhere to horse brush-border membranes. Purified K88ac pili when reacted with equine brush-border membranes inhibited to a great extent the adhesion of K88-positive E. coli. Similarly, K88-positive E. coli previously reacted with K88 antibody, did not attach to equine brush-border membranes. Oral inoculation of 4 newborn foals with strains of K88-positive enterotoxigenic E. coli, producing either heat-stable or heat-labile enterotoxin, caused diarrhoea in 1 animal.     

华东地区部分猪源大肠杆菌K88黏附素的生物学特性

近年来，从华东地区患腹泻仔猪中分离到一些表达K88菌毛的大肠杆菌，这些菌株只与K88a因子单抗反应，而不与b、c、d因子单抗反应。通过K88常规血清交叉吸收试验、SDS-PAGE、Western印迹，表明这些菌株不仅与K88ac参考菌株C83907制备的c因子血清反应，而且与以分离株SEC586制备且经K88ab、K88ac、K88ad参考菌株吸收后的血清也反应。对分离株SEC586、SEC464的K88主要亚单位结构基因faeG的克隆、测序，发现该基因由846对核苷酸组成，编码菌毛主要亚单位的262个氨基酸及21个氨基酸的信号肽，比国外报道的K88ac FaeG亚单位（263个氨基酸）少了1个氨基酸，比K88ab、K88ad（265个氨基酸）少了3个氨基酸。SEC586、SEC464菌株的FaeG亚单位氨基酸序列的同源性为97．7％，它们与K88ac的同源性为94．7％和96．2％；与K88ab的同源性为90．1％和91．2％；与K88ad的同源性为87．0％和88，6％。结果表明，新分离的K88ac大肠杆菌黏附素主要亚单位已发生了部分变异。     

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     

In vitro adhesion of K88ac+ Escherichia coli to Peyer''s patch and peripheral blood lymphocytes, buccal and rectal epithelial cells or intestinal epithelial brush borders of weaned pigs

Escherichia coli adhesion assays were conducted using isolated porcine peripheral blood lymphocytes, Peyer's patch lymphocytes, rectal epithelial cells or brush borders, buccal epithelial cells and brush borders from small intestinal epithelial cells. The cells and brush borders were tested for their ability to bind K88-piliated exterotoxigenic E. coliStrain M1823B (K88ac) and E. coli Strain 1476 (K-12, K88ac). Comparison of adhesive phenotypes of 37 weaned pigs as determined by the adhesion assay with small intestinal brush borders and the adherence of K88ac⁺ enterotoxigenic E. coli to peripheral blood lymphocytes, Peyer's patch lymphocytes and rectal epithelial cells or brush borders, revealed no correlation. In vitro adhesion of K88ac-bearing E. coli was always negative with buccal epithelial cells. K88ac strains varied in their ability to adhere to lymphocytes and rectale pithelial cells or brush borders, indicating that the mechanism of adherence is unrelated to K88-mediated adhesion observed in animals that had the receptors on small-intestinal epithelial-cell brush borders. The non-piliated control E. coli Strain 123 adhered to fresh peripheral blood lymphocytes, and less intensively to frozen-thawed peripheral blood lymphocytes or Peyer's patch lymphocytes. It was concluded that none of the cell types or brush borders, except small-intestinal epithelial-cell brush borders, could be used as targets for phenotyping pigs for the presence of the K88 receptors that have been associated with adhesion and colonization of K88⁺ enterotoxigenic E. coli in the porcine small intestine.     

In vitro adhesion of K88acEscherichia coli to Peyer's patch and peripheral blood lymphocytes, buccal and rectal epithelial cells or intestinal epithelial brush borders of weaned pigs

Escherichia coli adhesion assays were conducted using isolated porcine peripheral blood lymphocytes, Peyer's patch lymphocytes, rectal epithelial cells or brush borders, buccal epithelial cells and brush borders from small intestinal epithelial cells. The cells and brush borders were tested for their ability to bind K88-piliated exterotoxigenic E. coliStrain M1823B (K88ac) and E. coli Strain 1476 (K-12, K88ac). Comparison of adhesive phenotypes of 37 weaned pigs as determined by the adhesion assay with small intestinal brush borders and the adherence of K88ac⁺ enterotoxigenic E. coli to peripheral blood lymphocytes, Peyer's patch lymphocytes and rectal epithelial cells or brush borders, revealed no correlation. In vitro adhesion of K88ac-bearing E. coli was always negative with buccal epithelial cells. K88ac strains varied in their ability to adhere to lymphocytes and rectale pithelial cells or brush borders, indicating that the mechanism of adherence is unrelated to K88-mediated adhesion observed in animals that had the receptors on small-intestinal epithelial-cell brush borders. The non-piliated control E. coli Strain 123 adhered to fresh peripheral blood lymphocytes, and less intensively to frozen-thawed peripheral blood lymphocytes or Peyer's patch lymphocytes. It was concluded that none of the cell types or brush borders, except small-intestinal epithelial-cell brush borders, could be used as targets for phenotyping pigs for the presence of the K88 receptors that have been associated with adhesion and colonization of K88⁺ enterotoxigenic E. coli in the porcine small intestine.     

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.     

Experimental colibacillosis in gnotobiotic piglets exposed to 3 enterotoxigenic serotypes

Three strains of enterotoxigenic Escherichia coli (ETEC) (064:KSNT, K88ac; 020:KSNT, K88ac and 08:K85ab, K99) originally cultured from outbreaks of diarrhoea in piglets a few hours old, were administered orally to gnotobiotic piglets. There was a marked age-related difference in the clinical response to infection between the 3 strains although they all produced heat-stable toxin. All 3 strains produced severe clinical signs of depression, anorexia, vomiting, diarrhoea, followed by dehydration and death in one-day-old piglets. In piglets infected at 3 days of age the two K88+ ETEC caused diarrhoea and death but the K99+ ETEC induced moderate diarrhoea only. In piglets infected at 7 days of age, the 064 strain produced severe diarrhoea and death, and 020 strain caused mild diarrhoea in 3 of 6 piglets with one death while the 08 strain caused no illness. Pathological changes in the intestinal tract associated with these infections were minimal, or absent. Immunofluorescent staining with homologous hyperimmune sera demonstrated adherence of the 3 ETEC strains to the brush border of small intestinal epithelial cells. Fluorescing organisms were observed in all infected piglets irrespective of the severity of clinical signs but the degree and extent of colonisation varied with the age of the piglets and the infecting strain. This may explain the difference in clinical response between the 3 strains.     

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.     

Emigration of polymorphonuclear leucocytes into the intestinal lumen of the neonatal piglet in response to challenge with K88-positive Escherichia coli

The emigration of neutrophils from the blood of neonatal piglets into the intestinal lumen in response to a K88-positive strain of Escherichia coli was investigated. The pig herd used was of known genetic susceptibility to K88-positive E coli and had recently experienced an outbreak of neonatal diarrhoea. Neutrophil emigration depended on certain factors. Neutrophils emigrated into ligated loops in susceptible piglets sucking immune colostrum from susceptible dams but not into loops in colostrum deprived resistant piglets or piglets sucking non-immune colostrum from resistant dams. In susceptible, colostrum deprived piglets neutrophils in intestinal contents were only associated with severe lesions. Large numbers of neutrophils which appeared at several foci on the villi were observed in three of six resistant piglets that sucked immune colostrum from susceptible dams. It was concluded that neutrophil emigration into the intestinal lumen of piglets could occur in response to K88-positive E coli and resulted not from the presence or absence of the intestinal K88-receptor but from the ingestion of immune colostrum.     

大肠杆菌K88的黏附性与仔猪初生重、断奶重和日增重的关系

研究利用3个品种(大白、长白和松辽黑猪)共366头35日龄左右断奶仔猪的小肠样品与大肠杆菌K88ab、K88ac、K88ad进行黏附实验,并且利用SAS8.2对不同黏附类型仔猪的初生重、35日龄断奶重以及断奶前日增重进行了方差分析。结果表明:弱黏附归属不同时,其分析结果都是一致的,即黏附型仔猪与非黏附型仔猪的初生重、35日龄断奶重和断奶前日增重都没有显著差异(P>0.05)。     

Protection of the nursing pig against experimentally induced enteric colibacillosis by vaccination of dam with fimbrial antigens of E coli (K88, K99 and 987P)

Pregnant gilts were vaccinated with two doses of alhydrogel adsorbed fimbrial antigens of Escherichia coli (K88ab, K88ac, K99 and 987P) supplemented with beta toxoid of Clostridium perfringens type C. Their piglets, and piglets of nonvaccinated gilts, were subsequently orogastrically challenged with one or other of the four fimbrial types of enteropathogenic E coli. Some of the vaccinated animals were reinjected with a single dose of the vaccine during second gestation and their piglets, and piglets of non-vaccinated sows, were challenged the same way as were litters of gilts. Blood serum and colostra were examined for antibodies to the four fimbrial antigens of E coli and for antitoxin to beta toxin of C perfringens type C. It was found that: (1) a highly significant reduction in mortality and morbidity was achieved in vaccinated litters against all four challenge strains of E coli; (2) excretion of K88ab and K88ac but not of K99 and 987P challenge strains was significantly reduced; (3) revaccination of sows by a single dose of the vaccine during second gestation conferred complete protection against mortality and highly significant protection against morbidity; (4) no correlation was noted between colostral or seroagglutinins to fimbrial antigens of E coli and mortality rates in litters challenged with homologous fimbrial types of E coli, but good correlation was found between colostral precipitins to K88 antigens and mortality rates in litters; (5) antitoxin value in 97 per cent of colostrum of vaccinated sows was 10 iu equivalent of C perfringens type C toxin or more per ml of colostrum.     

仔猪黄痢K88-K99-987p-F41多价菌毛疫苗的制备及小鼠免疫试验

为了更好地预防仔猪黄痢,选取野生分离菌株HN2001（K88ab）、HN2002（K88ac）、HN2003（K88ad）、HN2004（K99）、HN2005（987p）和HN2006（F41）培养后用低温磁力搅拌法提取菌毛,制成5批多价菌毛混合油乳剂灭活苗。试验结果表明,5批多价灭活疫苗对小鼠的平均保护率达95．0％,为进一步进行本体动物试验提供了有价值的参考资料。     

Inheritance of K88-mediated adhesion of Escherichia coli to jejunal brush borders in pigs: A genetic analysis

The transmission and genetic organization of the adhesion of the serological variants of the K88 adhesin in the jejunum of the pig were investigated. The results of 28 matings of 5 boars with 15 sows are presented. On the basis of previous studies it has been accepted that the presence of specific receptor sites for K88ab and K88ac depends on a gene locus with 2 alleles S and s. The presence of additional receptor sites for K88ad is now presumed to depend on a separate locus with the alleles D and d. The expression of the alleles of the S and D loci is not always complete and is likely to be influenced by epistatic genes. Inhibition or modification of the expression of the receptor sites for K88 can result in intermediate phenotypes.     

大肠杆菌K_(88ac)与LT(A~-B~ )抗原基因质粒在猪霍乱沙门氏菌弱毒株中的表达

应用细菌质粒转化技术,将大肠杆菌K_(?)与LT(A~-B~ )抗原基因重组质粒转入猪霍乱沙门氏菌弱毒菌苗株中.对获得的其中8个转化子进行鉴定的结果表明,转化的细菌仍保持沙门氏菌的形态、生化及抗原特性,同时可稳定地表达K(?)和LT-B两种抗原.用微量间接血凝试验、抗甘露糖豚鼠红细胞凝集试验(MRHA)、ELISA等对转化菌表达的K(?)抗原进行了测定,用间接免疫溶血试验对其表达的LT-B抗原进行了测定.结果,这两种抗原在转化的细菌中均可高效表达.电镜下观察,转化的细菌在其表面形成菌毛样结构.这种转化细菌表现出猪霍乱沙门氏菌与产肠毒素性大肠杆菌的两种抗原特性,为这种双价工程菌苗的研制提供了有价值的候选菌株.本研究结果还表明,猪霍乱沙门氏菌弱毒菌苗株可作为基因转化的有效受体菌.     

In vitro inhibition of ETEC K88 adhesion by pea hulls and of LT enterotoxin binding by faba bean hulls

Enterotoxigenic Escherichia coli (ETEC) expressing K88 (F4) adhesins are associated with post‐weaning diarrhoea in piglets. Different grain fractions from pea (Pisum sativum) and faba bean (Vicia faba) were tested in vitro for their capacity to counteract aetiological factors, which contribute to the development of diarrhoea. In detail, adhesion of E. coli O149:K91:K88ac (ETEC K88ac) to grain legume products, intended to impair the colonization of the host, was studied as well as interference with receptor binding of the pathogen’s heat‐labile enterotoxin LT, intended to reduce toxin‐inflicted gut cell damage. When comparing different pea and faba bean products tested for their binding capacity of ETEC K88ac, especially pea hulls, but also whole pea meal, starch‐enriched and protein‐enriched pea meal, and digestion‐resistant pea hull and meal fractions showed a higher binding of ETEC K88ac than faba bean products. In contrast to the ETEC K88ac adhesion results, bean hulls proved more effective than pea hulls in preventing GM1 receptor binding of LT. Previous small intestinal segment perfusion experiments we performed with ETEC K88ac‐challenged piglets indicated that both pea and bean hulls have the potential for successful application in diarrhoea prophylaxis and treatment, which is in agreement with and refined by our detection of their different modes of functioning.     

Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic Escherichia coli strain that expresses the same adhesion and enterotoxins

Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin.     

Effect of experimentally-induced villus atrophy on adhesion of K88ac-positive Escherichia coli in just-weaned piglets

Three- to four-week-old, just-weaned piglets were infected with transmissible gastroenteritis (TGE) virus and the next day with K88ac+ enterotoxigenic Escherichia coli (ETEC). Histological examination of caudal jejunum and ileum of piglets killed 2-3 days after virus challenge (1-2 days after ETEC infection) revealed severe villus atrophy especially in the jejunum compared with controls (P less than 0.05). Four-5 days after TGE virus infection villus length increased and after 7 days it was near normal. Villi scraped from jejunal and ileal mucosa of the piglets were incubated in vitro with K88ac+ E. coli and the number of bacteria adhering to 250 micron villus brush border was counted. Attachment of bacteria to villi of piglets killed 2-3 days after TGE virus infection was significantly decreased in comparison with adhesion to villi of non-infected piglets or of piglets killed 7 days after the virus infection. Correlation between in vitro adhesion and villus height was 0.6649 (P less than 0.001). The results suggest that the experimentally-induced villus atrophy was attended with a temporarily diminished susceptibility of villus enterocytes to adhesion of K88ac+ E. coli.     

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.     

Metabolic changes in red cells in response to adhesion of porcine K88 fimbriated Escherichia coli

Red cells glycolytic enzymes attached and nonattached to K88⁺ Escherichia coli were assayed. Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase activities, were measured. E. coli with K88ab fimbriae, E. coli with K88ac fimbriae, and isolated K88ab fimbriae were investigated for their effect on the above enzymes. Different changes were obtained with K88ab + bacteria compared with K88ac + bacteria. Purified fimbriae gave a third set of responses.