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.     

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.     

Prevalence of four enterotoxin (STaP, STaH, STb, and LT) and four adhesin subunit (K99, K88, 987P, and F41) genes among Escherichia coli isolates from cattle

Colony hybridizations with DNA probes for 3 heat-stable (STaP, STaH, and STb) enterotoxins and 1 heat-labile (LT) enterotoxin and for 4 adhesins (K99, F41, K88, 987P) were performed on 870 Escherichia coli isolates to determine pathotypes prevalent among enterotoxigenic E coli (ETEC) isolated from cattle in Belgium. One hundred thirty-two E coli isolates (15.2%) hybridized with probes STaP, K99, and/or F41. The 5 other probes were not hybridized by E coli isolates. Therefore, only STaP enterotoxin and K99 and F41 adhesins were virulence factors of ETEC isolated from cattle. Two major pathotypes accounted for 95% of the ETEC: STaP+K99+F41+ (67.4%) and STaP+K99+ (27.3%). The last 5% of probe-positive isolates had STaP+, STaP+F41+, or K99+F41+ minor pathotypes. Of 12 American ETEC isolates also assayed, 7 were positive with STb and/or 987P probes (pathotypes STaP+STb+, STaP+ 987P+, or STaP+STb+987P+) and may be porcine- rather than bovine-specific enteropathogens. The remaining 5 American ETEC isolates belonged to 3 minor pathotypes (STaP+, STaP+F41+, and K99+F41+) also found among Belgian E coli isolates. Such isolates may be derivatives of STaP+K99+F41+ or STaP+K99+ ETEC after in vivo or in vitro loss of virulence genes and/or non-ETEC isolates, which have acquired virulence genes by in vivo transfer.     

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.     

Characterization and purification of the F17 adhesin on the surface of bovine enteropathogenic and septicemic Escherichia coli

The F17 antigen from bovine enterotoxigenic Escherichia coli strain (E coli 25KHO9), which adhered to calf intestinal villi, was isolated. An enterotoxin-negative derivative (25KHO9st) was used for further studies. Using an immunogold-labeling technique, the F17 antigen was characterized as a fimbrial protein. Pure fimbriae with a subunit molecular weight of 20,000 were obtained by homogenization and use of a sucrose gradient. The adhesion of E coli 25KHO9st was mediated by the F17 fimbriae, as both F17 antibodies and F17 protein blocked the adhesion of the strain 25KHO9st. The F17 fimbriae were serologically distinct from K88, K99, F41, and 987P fimbriae and did not agglutinate bovine, ovine, guinea pig, human, or chicken erythrocytes. Peptide fingerprint analysis revealed F17 and F(Y) adhesins to be homologous, if not identical.     

Prevalence of virulence genes in Escherichia coli strains recently isolated from young pigs with diarrhea in the US

Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning diarrhea (PWD) is economically one of the most important diseases for the swine industry. Porcine ETEC strains typically express K88 or F18 fimbria and heat-labile (LT) and/or heat-stable (STa, STb) enterotoxins. However, recent studies indicate that EAST1 toxin, adhesin involved in diffuse adherence (AIDA-I) and porcine attaching and effacing-associated factor (paa) may also be expressed by ETEC strains associated with diarrhea. To better understand the virulence factors of E. coli strains that cause PWD, we applied PCR to screen for K88, F18, F41, 987P and K99 fimbrial genes; LT, STa, STb, Stx2e and EAST1 toxic genes; and AIDA-I, paa and EAE adhesin genes in E. coli strains recently isolated from young pigs with PWD in the US. Of 304 E. coli isolates from diarrheic pigs submitted for testing, 175 (57.6%) strains possessed fimbrial genes: K88 (64.6%), F18 (34.3%), F41 (0.57%), K99 (0.57%), 987P (0); toxin genes: LT (57.7%), STb (72.6%), STa (27.4%), STx2e (17.4%), EAST1 (35%); and adhesin genes: AIDA-I (26.9%), paa (60%), EAE (1.1%). All toxin genes except the EAST1 toxin gene, were almost exclusively associated with K88+ or F18+ isolates, and most of these isolates carried multiple toxin genes. The non-fimbrial adhesin paa was found present in over half of the K88+ isolates. A total of 129 (42%) isolates carried no fimbrial genes, including 66 (21.7%) isolates that did not have any of the above virulence genes. These results suggest a broad array of virulence genes associated with PWD in 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.     

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.     

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.     

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)     

Evaluation of a monoclonal antibody to the K99 fimbrial adhesin produced by Escherichia coli enterotoxigenic for calves, lambs and piglets

All the K99+ Escherichia coli grown at 37 degrees C stained strongly with a peroxidase labelled K99 monoclonal antibody using a direct immunoperoxidase staining procedure. There was no reaction when these bacteria were cultured at 18 degrees C or when K99- E coli were grown at either temperature. The binding of the monoclonal antibody to K99 antigen was inhibited by OK antisera to heterologous K99+ E coli but OK antisera to E coli producing adhesins other than K99 were without effect. Using the slide agglutination test the reactions of the monoclonal antibody were identical to those of a polyclonal antiserum to K99 when both were used in parallel to examine 100 K99+ E coli from at least 10 somatic O groups and 1308 K99+ E coli from at least 82 different somatic O groups submitted for routine serological typing in England or the, USA. The monoclonal antibody reacted with K99+ E coli in cryostat sections of the ileum from a piglet infected with E coli strain B44 (O9: K30, K99, F41) but there was no reaction with similar material from piglets infected by E coli strains 1751 (O101: F41), X177/81 (O9: K103, 987P) or Abbotstown (O149: K91, K88ac).     

A comparison of histopathological changes in calves associated with K99- and K99+ strains of enterotoxigenic Escherichia coli.

Enterotoxigenic colibacillosis was experimentally produced in four colostrum-deprived calves given 10(10) Escherichia coli strain 210 (serotype 09+:K30+:K99-:F41-:H-) orally and the histopathological changes compared to those seen in colostrum-fed calves infected in an earlier study with strain B44 (serotype 09+:K30+:K99+:F41+:H-). Escherichia coli strain 210 caused diarrhea, atrophic villi with cuboidal epithelium, and focal accumulations of a few neutrophils in the dome villi above Peyer's patches but neither the clinical nor the histopathological changes were as pronounced as with strain B44. The extent and distribution of adherence to the mucosal surface differed between the two strains. Strain B44 adhered as a continuous layer over most of the absorptive epithelial surface of both the jejunum and ileum. Adherence of strain 210 was restricted to the ileum and the bacteria often adhered focally in "clumps" rather than as a continuous layer, especially on the distal half of the villous surface.     

Nine DNA probes for detection of toxin and adhesin genes in Escherichia coli isolated from diarrhoeal disease in animals

DNA gene probes specific for genes encoding heat labile enterotoxin (LTI), heat stable enterotoxins (STIa, STII), vero cytotoxins (VT1, VT2), and adhesins K88 (F4), K99(F5), F41 and 987P(F6) were used to examine 873 isolates of E. coli from cases of diarrhoea (680 from pigs, 187 from cattle and six from sheep). A total of 188 were toxin gene positive and of these 84 belonged to the classical ETEC serogroups. Of the other 104 toxin gene positive strains, 80 hybridized with the VT2 probe of which 34 were from cases of porcine post-weaning diarrhoea belonging to serogroup 0138:K81 and 22 were untypable strains from cattle.     

Enhanced expression of 987P (F6) fimbrial adhesin by cultured enterotoxigenic Escherichia coli.

The expression of the 987P fimbrial adhesin by strains of enterotoxigenic Escherichia coli was enhanced and, in some cases, restored by passaging the organisms through Craigie's tubes. In contrast, the fimbrial adhesins K99, K88 and F41 were not optimally expressed in this medium. The results suggest that Craigie's tubes should be used for the optimum expression of 987P.     

卵黄抗体对ETEC粘附仔猪小肠上皮细胞的体外抑制作用研究

采用热抽提法提取 4种肠毒素性大肠杆菌菌毛蛋白 :K88、K99、F41和 987p。分别制成单价或多价的菌毛蛋白白油佐剂抗原 ,对产蛋鸡进行胸部肌肉分点注射免疫 ,初免后 2周加强免疫 1次。收集高效价卵黄抗体。用所获得各卵黄抗体对体外分离的初生仔猪小肠上皮细胞进行体外粘附抑制试验。结果表明 ,各种菌毛卵黄抗体均能特异地显著抑制相应大肠杆菌对仔猪上皮细胞的粘附 ,而对其他血清型大肠杆菌对肠上皮细胞的粘附无抑制作用     

Development of vaccines for bacterial diseases using recombinant DNA technology

A vaccine was prepared using recombinant DNA techniques to prevent fatal enterotoxigenic Escherichia coli diarrhea in swine. The product, which is a subunit vaccine, was prepared by mechanical and chemical removal of pilus adhesins from the surface of genetically engineered strains of E. coli. The vaccine contains the pilus adhesins K88, K99, and 987P plus an adjuvant. The genes responsible for production of K88 and K99 were separately cloned into the multicopy vector pBR322. K88 was found to be encoded on a 7.6-kilobase HindIII-EcoRI fragment, and K99 was found to be encoded on a 7.15-kilobase BamHI fragment. Strains containing the recombinant plasmid for K99 produced up to ten times more K99 than strains containing the wild-type plasmid. Vaccination of pregnant pigs with the vaccine led to production of pilus-adhesin-specific antibodies that were transferred to the piglets in colostrum and milk. Pilus-adhesin-specific antibodies neutralized the adhesiveness of the pili on enterotoxigenic E. coli, thus preventing attachment, colonization, and disease. Mortality of pigs in litters from vaccinated pigs due to experimentally induced enterotoxigenic E. coli diarrhea was reduced 10-to-20-fold (depending upon the challenge strain), and the incidence, severity, and duration of diarrhea were also reduced.     

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.     

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.     

Enterotoxigenic Escherichia coli (ETEC) in farm animals.

Animal diseases due to enterotoxigenic Escherichia coli (ETEC) typically appear as severe watery diarrhoea during the first few days of life (also a few days after weaning in pigs). ETEC adhere to the small intestinal microvilli without inducing morphological lesions and produce enterotoxins acting locally on enterocytes. This action results in the hypersecretion (of water and electrolytes) and reduced absorption. Adhesins and toxins are the two prominent virulence attributes of ETEC and the level of knowledge of these factors determines the chances for successful prevention and therapy of the disease. For animal ETEC the most common adhesins are the fimbriae (pili) on the surface: F4(K88), F5(K99), F6(987P), F41, F42, F165, F17 and F18. Enterotoxins (extracellular proteins or peptides) of animal ETEC are classified as heat-labile (LT) and heat-stable (ST) enterotoxins with further subdivisions (LTh-I, LTp-I, LTIIa, LTIIb, STaH, STaP, STb) according to antigenic and biological differences. Fimbriae and LT enterotoxins are made up of large molecular weight proteins which facilitate their utilisation in vaccines and their detection using immunodiagnostic systems. The adhesive fimbriae and enterotoxins of animal ETEC are plasmid determined (except F41 and F17). Virulence gene probes (DNA hybridisation, PCR) are specific and sensitive diagnostic and epidemiologic tools for the detection of ETEC. Based on genetic typing, the ETEC, in spite of limited serogroups, seem to represent a population of E. coli with a diverse genetic background.     

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.