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.     

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.     

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.     

Relationship between virulence and adherence of various enterotoxigenic Escherichia coli strains to isolated intestinal epithelial cells from Chinese Meishan and European large white pigs

In vitro adherence to intestinal epithelial cells by enterotoxigenic Escherichia coli strains bearing K88, K99, F41, or 987P adhesins and of their variants not bearing adhesins (K88-, K99-, or F41-) was investigated in European Large White and Chinese Meishan pigs. Possible relationship between adherence and virulence was also examined. The K88-positive (K88+) strain strongly adhered to intestinal epithelial cells from 26 of 28 Large White pigs. This strain had previously been found to be highly virulent for Large White pigs. The only surviving pig was of nonadherent phenotype, and cells from 4 dehydrated moribund pigs had strong adherence. By contrast, the same K88+ strain found previously to have little pathogenicity for Meishan pigs adhered with variable intensity to cells from 17 of 23 Meishan pigs; correlation was not evident between adherence and virulence. The K99+ F41+ strain of porcine origin and the F41+ strain generally adhered strongly to cells from 24 and 23 Meishan pigs, respectively, and to cells from 25 of 26 Large White pigs. Correlation was not found between adherence and virulence for the 2 strains. A K99+ F41+ strain of bovine origin adhered to cells from 20 of 22 Meishan and 22 of 23 Large White pigs, and a K99- F41+ variant adhered to cells from 19 of 23 Meishan and 23 of 24 Large White pigs. The adhesin-negative variants never adhered to intestinal epithelial cells. Strain 987 known not to readily produce 987P adhesin after in vitro growth never adhered to cells during the test.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.     

Evaluation of monoclonal antibodies to K88, K99, F41 and 987P fimbrial adhesins for the detection of porcine enterotoxigenic Escherichia coli in paraffin-wax tissue sections

A panel of monoclonal antibodies against fimbrial adhesins of porcine enterotoxigenic Escherichia coli were evaluated for the detection of enteric colibacillosis in paraffin-wax embedded sections of piglet small intestine. Using the immunoperoxidase technique, monoclonal antibodies were used to detect epitopes on the K99 adhesin and on the a and c regions of the K88 adhesin. However, monoclonal antibodies to the F41 and 987P adhesins failed to react in sections with organisms colonising the intestine of gnotobiotic piglets monoinfected with strains bearing those adhesins, whereas corresponding polyclonal antisera gave positive results. In contrast to apparent expression of all K99 organisms, only a proportion of organisms were identified by monoclonal or polyclonal antibodies as expressing K88. In some instances, failure of immunostaining was attributed to prolonged storage of tissue in formalin.     

Preliminary characterization of Escherichia coli isolated from pigs with diarrhoea in Venezuela

The aetiology of neonatal porcine diarrhoea was studied in 15 different herds located in the north-western region of Venezuela. Of 56 strains of Escherichia coli analyzed, 16 (28.6%) were shown to produce heat-stable (STa) enterotoxin, as detected by infant mouse assay. Only four of these STa+ isolates also possessed the K88 pilus antigen, two were 987P+ and none possessed the K99 antigen, leaving 10 STa+ samples in which no pilus antigen was identified. Among the 40 STa negative samples were six K88+ specimens, one K99+, four 987P+, one which reacted as K88+ + K99+ and one K88+ + 987P+. Considering as pathogenic any strain showing at least one of the characters studied, pathogenic E. coli were detected with an overall frequency of 42.9%, being more prevalent during the second week of life. An electrophoretic analysis of the plasmid content of the field isolates of E. coli, revealed the presence of numerous species of extrachromosomal DNA, although no direction association could be made between a particular plasmid and any of the pathogenic characteristics identified. Results of Southern blot analysis indicate that the STa enterotoxin was preferentially encoded within an endemic plasmid of 4.9 Md. Other plasmids present in the E. coli isolates could be related to antibiotic resistance. With the exception of one strain, all E. coli isolates were resistant to more than one of the nine drugs tested; multiresistant E. coli were frequently isolated, including four strains which were resistant to seven antibiotics.     

Evaluation of monoclonal antibodies to K88, K99, F41 and 987P fimbrial adhesins for the detection of porcine enterotoxigenic Escherichia coli in paraffin-wax tissue sections

A panel of monoclonal antibodies against fimbrial adhesins of porcine enterotoxigenic Escherichia coli were evaluated for the detection of enteric colibacillosis in paraffin-wax embedded sections of piglet small intestine. Using the immunoperoxidase technique, monoclonal antibodies were used to detect epitopes on the K99 adhesin and on the a and c regions of the K88 adhesin. However, monoclonal antibodies to the F41 and 987P adhesins failed to react in sections with organisms colonising the intestine of gnotobiotic piglets monoinfected with strains bearing those adhesins, whereas corresponding polyclonal antisera gave positive results. In contrast to apparent expression of all K99 organisms, only a proportion of organisms were identified by monoclonal or polyclonal antibodies as expressing K88. In some instances, failure of immunostaining was attributed to prolonged storage of tissue in formalin.     

Linkage of genes for heat-stable enterotoxin, drug resistance, K99 antigen, and colicin in bovine and porcine strains of enterotoxigenic Escherichia coli

Linkages among genes for production of Escherichia coli heat-stable enterotoxin (ST), drug resistance, K99 antigen, and colicin were investigated in 2 bovine and 3 porcine strains of enterotoxigenic E coli. In conjugation experiments, all 5 isolates transferred enterotoxigenicity and the ability to produce K99; 4 of the 5 isolates also transferred antibiotic resistance markers, and 3 colicinogenic strains transmitted the ability to produce colicin. In 2 of the 3 colicin-producing strains, the genes for colicin were located along with those for K99 and ST on a single plasmid. One of these 2 strains transferred the genes for tetracycline resistance and production of both mouse-active ST (STa) and mouse-inactive ST, whereas the other transmitted the gene(s) for STa only. Transformation studies with the 3rd strain revealed that the K99 determinant resided on a 22-megadalton (Mdal)R plasmid, and that STa and colicin production were on a 65-Mdal plasmid. Analysis of the plasmids from the transformation experiments revealed that the larger plasmid was conjugative and the smaller plasmid was nonconjugative; stable cointegrate formation occurred between these 2 plasmids. Genetic information coding for the production of STa and K99 were also present on a single plasmid of approximately 80 Mdal in both noncolicinogenic strains.     

对FC株猪源性肠毒素型大肠杆菌致病因子的研究

FC菌株是一株从腹泻仔猪粪便中分离的肠毒素型大肠杆菌(Enterotoxigenic E.coli,ETEC)。在MRHA反应中,本菌能凝集人O型、豚鼠、马、绵羊、牛、鸡和兔的红细胞,对人O型和豚鼠红细胞有很高的血凝性,血抗K88和K99血清不能抑制其对豚鼠和绵羊红细胞的血凝。在体外小肠上皮细胞吸附试验中,本菌对仔猪小肠上皮细胞具有强烈的吸附作用;透射电镜和扫描电镜观察证实了FC株菌除表面具有一种纤毛样结构外,还能定居在仔猪小肠段。血清学试验结果表明,本菌的O抗原属于O101。K88和987P两种抗血清均不能凝集本菌,而K99和F41抗血清均可凝集。对纯化的FC株菌粘着素抗原作等电聚焦和聚丙烯酰胺凝胶电泳分析,结果表明,该菌的粘着素是由等电点分别为4.61和9.78,分子量分别为29500和17500的两种蛋白质抗原所组成。此外,用乳鼠胃内投服试验和兔肠结扎试验证明,该菌只产生热稳定肠毒素。总之,本菌是一株能产生ST的K99,F41的肠毒素型大肠杆菌。     

Prevalence of fimbrial antigens and enterotoxins in nonclassical serogroups of Escherichia coli isolated from newborn pigs with diarrhea

Ninety-nine nonclassical serogroups isolated from newborn pigs with neonatal diarrhea were tested for fimbrial antigens F4(K88), F5(K99), F6(987P), F41, and F165, and for heat-labile enterotoxin, heat-stable enterotoxin a (STa), heat-stable enterotoxin b, verocytotoxin, and cytolethal-distending toxin. Thirty-two strains, belonging mostly to serogroups O64:K"V142,", O9:K103, and O20:K101, were F5-positive and usually produced STa, although 5 strains produced only heat-stable enterotoxin b. Fifteen strains, belonging mostly to serogroups O64:K"V142" and O20:K101, were F41 positive and usually produced STa. Twenty-three stains, belonging mostly to serogroups O64:K"V142," O9:K103, and O20:K101, were F6-positive and usually produced STa. Several strains produced more than one fimbrial antigen, either F5 and F41, F5 and F6, F6 and F41, F6 and F165, or F5, F6, F41, and F165. None of the strains produced heat-labile enterotoxin, verocytotoxin, or cytolethal-distending toxin. The indirect immunofluorescence test was much more sensitive than was the slide agglutination test for the detection of each of the fimbrial antigens F5, F6, F41, and F165 on strains grown in vitro. The production of F6 by certain strains for which only a low proportion of cells were F6-positive in vitro, as demonstrated by immunofluorescence, was confirmed by experimental infection of newborn pigs.     

Requirement for capsular antigen KX105 and fimbrial antigen CS1541 in the pathogenicity of porcine enterotoxigenic Escherichia coli O8:KX105 strains.

The requirement for capsular antigen KX105 and fimbrial antigen CS1541 in the pathogenicity of porcine enterotoxigenic Escherichia coli O8:KX105 strains lacking the colonization factor antigens K88, K99, 987P and F41 was investigated using two encapsulated strains and their acapsular variants, one of which produced the fimbrial antigen CS1541 in vitro. None of the strains adhered in vitro to enterocytes isolated from newborn colostrum-deprived piglets. All of the strains caused diarrhea in orally infected, hysterotomy-derived, colostrum-deprived piglets although a great variability in the clinical response of the piglets was observed. Colonization of the small intestine of infected piglets by these strains was only moderate and no differences in the ability to colonize the small intestine was noted between the strains. All of the strains reacted in the indirect fluorescent antibody test with both CS1541 and 987P antisera when applied to organisms in the intestines of infected piglets. A control strain expressing the 987P fimbrial adhesin also reacted with the CS1541 antiserum applied to organisms in the intestines of an infected piglet. It was concluded that capsular antigen KX105 was not essential for intestinal colonization and production of diarrhea in hysterotomy-derived colostrum-deprived pigs, and that fimbrial antigen CS1541 does not promote in vitro adherence to enterocyte brush borders but could be important in bacterial colonization in vivo.     

仔猪大肠杆菌病K88-K99-987P-F41四价亚单位疫苗的研制Ⅳ.疫苗生产与免疫效力试验

选用4株带有K88、K99、987P、F41粘附素抗原的产肠毒素性大肠杆菌,分别接种于BBL、Minca、Slanetz和Minca培养基进行培养,将培养物加热提取粘附素抗原后,加入油佐剂制成四价亚单位疫苗。经成品检验合格后分别免疫小鼠和怀孕母猪,同时监测怀孕母猪抗体。最后1次免疫后15 d,分别用同源ETEC确定的攻毒剂量攻击。结果显示,经2次免疫后,K88、K99、987P、F41对小鼠的免疫保护率与1次免疫没有显著差异(P>0.05);对仔猪,1次免疫跟2次免疫均可显著地降低腹泻指数(P<0.05),2次免疫与1次免疫没有显著差异(P>0.05)。怀孕母猪免疫1周后(产前23 d)抗体开始上升,第2周(产前16 d)达到高峰。2次免疫后抗体迅速回升,第4周(产前2 d)达到最高峰,产后2 d抗体大幅度下跌,几近免疫前水平。二免母猪所产仔猪发病率明显低于一免母猪(P<0.05)。攻毒保护试验和抗体消长规律的结果表明,制备的仔猪大肠杆菌病K88-K99-987P-F41四价亚单位疫苗具有良好的免疫原性,能有效预防仔猪大肠杆菌病的发生。     

DNA probes for the detection of toxin genes in Escherichia coli isolated from diarrhoeal disease in cattle and pigs

DNA gene probes specific for genes coding for heat labile toxin (LT), heat stable toxins (STpa, STpb) and Vero-cell toxins (VT1, VT2) were used to examine 1031 diarrhoeal disease isolates of E. coli (345 from cattle and 686 from pigs). Of the bovine strains, 60 hybridized with the STpa probe and most possessed the K99 (F5) or F41 adhesin. Five bovine strains possessed STpb genes and five either VT1 or VT2 genes. Of the porcine strains, 245 hybridized with one or more gene probes. Of 160 K88 (F4) positive strains, 133 possessed both LT and STpb genes, whilst 17 possessed LT or STpb or STpa alone or in combination. Ten K88 strains did not possess toxin genes. Isolates bearing the K99 (F5) adhesion possessed either STpa, STpb and VT2 genes alone or in combination; in one isolate only the LT gene was detected. Isolates belonging to serogroup 0138:K81 were more heterogeneous as to their toxin genes; of the 60 strains, fourteen carried only VT2 genes, thirty-two carried VT2, STpa and STpb genes, one carried LT, VT2, STpa and STpb genes, two carried STpb gene, four carried STpa and STpb genes, one carried LT and VT2 genes, two carried LT and STpa genes, whilst four carried none. Twenty-four percent of all toxigenic strains apparently did not possess adhesins.     

Prevention of colibacillosis in neonatal swine with a 4-pilus E coli bacterin

Of 12 pregnant swine (vaccinates) given a 4-pilus enterotoxigenic E coli (ETEC) bacterin (K88, K99, 987P, F41), all developed comparable or significantly higher serum and colostral antibody levels than those of 8 pregnant swine (controls) given a 3-pilus ETEC bacterin (K88, K99, 987P). When piglets were challenged with an ETEC strain bearing the F41 antigen, those from vaccinates had significantly lower mortality, less scours, less severe clinical signs and better weight gain than those from controls.     

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.     

Enterotoxigenic Escherichia coli in the jejunum of piglets with neonatal diarrhea

Thirty-two Escherichia coli colonies were taken from the primary step of cultivation of the jejunal contents of each of 10 dead piglets which had suffered from diarrhea. The organisms of each colony were examined for the presence of adhesion fimbria (F4 (K88) and F5 (K99)), production of heat-stable and heat-labile enterotoxin and of colicins.The presence of heat-labile enterotoxin in the intestinal content of the necropsied pigs was also tested, and results correlated with enterotoxin production of the isolated E. coli strains. In all but 3 pigs, 50–80 % of the E. coli strains were found to produce one or both of the enterotoxins and to possess the F4 of the F5 antigen. All bacteria producing both heat-labile and heat-stable enterotoxin proved to belong toi O group 149 and to possess the F4 antigen. Strains from 1 pig belonged to O group 64 and possessed the F5 antigen; these bacteria produced heat-stable enterotoxin only. Most of the enterotoxin-producing E. coli also produced colicins.After each subcultivation, the strains produced less heat-labile enterotoxin, some becoming negative when assayed.     

共表达K88/987p菌毛产肠毒素性大肠杆菌的分离鉴定及其卵黄抗体的制备

为制备针对一些大型养猪场产肠毒素性大肠杆菌(enterotrxigenic Escherichia coli,ETEC)分离株的特异性卵黄抗体(egg yolk immunoglobulin,IgY),试验对从这些养殖场分离的ETEC分离株菌毛基因类型进行了PCR鉴定,纯化该分离株的菌毛蛋白免疫蛋鸡制备IgY,对该IgY的效价、特异性和体外抑菌效果进行了检测。结果表明,该分离株具有K88和987p 两种菌毛基因,纯化后的分离株菌毛具有较强的免疫原性,经3次免疫后产生的IgY对K88和987p全菌和菌毛的效价可达到1:64 000,分离株菌毛IgY能特异地与K88和987p反应,与K99、F41无交叉反应,5 mg/mL分离株菌毛IgY在体外具有很好的抑菌效果。     

Characterization of a new fimbrial antigen present in Escherichia coli strains isolated from calves.

Thirteen Escherichia coli strains isolated from calves with diarrhoea, supposed to carry a common antigen were examined for their hemagglutinating activity and compared by bacterial agglutination, double diffusion in two dimensions and by crossed immunoelectrophoresis (CIE). Two of the strains were examined also in the electron microscope. Most of the strains agglutinated red blood cells of horse, ox, guinea pig and chicken, of which the agglutination of ox erythrocytes was mannose-resistant (MRHA). None of the strains agglutinated human erythrocytes. All strains with MRHA of ox red blood cells, regardless to their O:K:H antigens could be agglutinated in unabsorbed or absorbed antisera produced against cultures C1209 (020:K-:H9) and C1213 (09:K36:H-) when live cells as antigens were used. None of these sera agglutinated reference strains carrying K88, K99, 987P, F41 or FY (Att25) antigen respectively. By the double gel diffusion test and by CIE in extracts (60 degrees C) of the strains a common heat labile antigen, responsible for the MRHA of ox red blood cells was identified. Electron microscopy revealed that this common antigen was represented by thin, long, hair-like fimbriae on cells of E. coli C1213, and that specific homologous antibodies attached to these fimbriae.