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1.
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.  相似文献   

2.
A comprehensive study of 223 Escherichia coli isolates from pigs with colibacillosis included determination of O serogroups, detection of heat-labile enterotoxin, heat-stable enterotoxin (STa and STb), and identification of K88, K99, 987-P, F-41, and type 1 fimbriae. The incidence of the various E coli types among isolates of pigs of different ages was also determined. Escherichia coli bearing K88 fimbriae accounted for 48% of all isolates studied, were most often of serogroup O157, O149, or O8, and usually produced labile toxin alone or in combination with STa or STb. These E coli were commonly isolated from pigs in each age group studied (0 to 5 days, 6 to 10 days, 11 to 24 days, and greater than 24 days). Escherichia coli bearing 987-P accounted for 30% of the isolates, were most often of serogroup O141 or O20, and usually produced STa. Escherichia coli bearing K99 accounted for 13% of the isolates, usually were of serogroup O101 or O8, and almost always produced STa. Escherichia coli bearing 987-P or K99 were most often isolated from pigs less than 6 days of age. Fimbriae F-41, when identified, were usually on E coli of serotype O101:K99. Although infrequently found, type 1 fimbriae were on E coli of most of the serogroups identified in this study.  相似文献   

3.
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.  相似文献   

4.
Fourteen strains of Escherichia coli of serogroups characteristic of porcine class 2 enterotoxigenic E. coli isolated from pigs or calves were selected for genetic studies. The strains were examined for their ability to cotransfer a number of plasmid-mediated properties during conjugation with E. coli K-12. These properties were antibiotic resistance, and the production of heat-stable enterotoxin, the K99 antigen and colicin and the ability to ferment raffinose. Distinction was made between the two types of heat-stable enterotoxin, STa and STb. All 14 strains were antibiotic resistant and 11 of them cotransferred antibiotic resistance and heat-stable enterotoxin. One strain which transferred heat-stable enterotoxin also transferred the raffinose gene. Among six K99-positive strains which transferred heat-stable enterotoxin, five always cotransferred K99. Three strains had 100% cotransfer of colicin as well as heat-stable enterotoxin and K99. Drug resistance determinants were cotransferred at high frequency with heat-stable enterotoxin for six of eight multiple drug resistant enterotoxigenic E. coli. A 100% cotransfer of combinations of heat-stable enterotoxin, K99, colicin and antibiotic resistance was often associated with a single plasmid band on agarose gel electrophoresis. For some strains, the genes for STa and STb were on the same plasmid and for others they were on separate plasmids. The enterotoxin plasmids ranged in size from 5.2 to 85 Mdal. Heterogeneity in molecular size occurred among enterotoxin plasmids in E. coli of the same serogroup and recovered from the same animal host species.  相似文献   

5.
Lymph node cells from calves immunized with purified pilus antigen of K99+ enterotoxigenic E. coli (ETEC) were fused with mouse myeloma (NSO) cells, and with non-Ig producing mouse/calf hybridomas or with a bovine Ig-producing mouse/calf/calf secondary hybridoma. Lines secreting bovine monoclonal IgG1 specific for K99 pilus antigen in an ELISA were obtained in each case. The two lines derived from xenohybridoma fusion partners have been secreting anti-K99 bovine monoclonal antibody for over one year in continual passage. None of the antibodies cross-reacted with other pilus types including K88, CFAI, CFAII, 987P or CP; they all inhibited agglutination of horse RBC (which have a K99 receptor) in the presence of K99 antigen; they showed positive fluorescence in an indirect binding assay on K99+ ETEC and inhibited K99+ ETEC adhesion to piglet enterocytes. These antibodies have potential prophylactic and therapeutic use in control and treatment of diarrhoea.  相似文献   

6.
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.  相似文献   

7.
A survey was undertaken in piggeries in the Bogor and Jakarta Capital Territory areas to identify the antigens associated with enterotoxigenic Escherichia coli (E. coli). Rectal swab samples were collected from 65 normal piglets and from 858 with diarrhoea. Fimbrial and O-antigens were determined by agglutination tests. The 987P antigen was only associated with non-haemolygic E. coli in colonies grown for 3-10 days in tryptic soy broth. Organisms which possessed 987P antigen were isolated from 56.4% of piglets with diarrhoea and from 10.8% of normal piglets. Most cases of diarrhoea that were associated with E. coli 987P occurred within the first 3 weeks of life. Multiple infections occurred in 13.4% of cases and were associated with E. coli K88 in eight cases (1.7%) K99 in 26 cases (5.4%) and F41 in 31 cases (6.4%). Of 959 isolates of E. coli 987P, 80.7% were O-group 20, 13% were O-group 9 and 0.5% were O-group 141 with 5.7% being non-typable. Heat stable toxin was produced by all five E. coli 987P isolates tested.  相似文献   

8.
Pregnant gilts were vaccinated orally with Escherichia coli that produced pilus antigens K99 or 987P. The vaccines were live or dead enterotoxigenic E coli (ETEC) or a liver rough non-ETEC strain which has little ability to colonize pig intestine. Pigs born to the gilts were challenge exposed orally with K99+ or 987P+ ETEC, which did not produce heat-labile enterotoxin or flagella and which produced somatic and capsular antigens different from those of the vaccine strains. Control gilts had low titers of serum and colostral antibodies against pilus antigens, and their suckling pigs frequently had fatal diarrhea after challenge exposure. Serum antibody titers against pilus antigens of the vaccine strains increased in the gilts after vaccination with liver ETEC, and the colostral antibody titers of these gilts were higher than those of controls. Pigs suckling such vaccinated gilts were more resistant than controls to challenge strains were of different pilus types, and it could not be attributed to enterotoxin neutralization by colostrum. In contrast to the live ETEC vaccines given to the pregnant gilts, the liver rough non-ETEC and dead ETEC vaccines stimulated little or no production of antibody against pilu, and the pigs born of these vaccinated gilts remained highly susceptible to challenge exposure. The results support the hypothesis that pilu can be protective antigens in oral ETEC vaccines. It was indicated that in the system reported, protection depended on living bacteria for the production of pilus antigens in vivo or for the transport of pilus antigens across intestinal epithelium.  相似文献   

9.
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.  相似文献   

10.
Direct fluorescent antibody tests were used to detect Escherichia coli possessing K88 and 987P antigens. Identification of bacteria was accomplished on suspensions of organisms from clinical isolates, on frozen sections and impression smears from small intestine and on faecal smears. This assay makes possible the rapid identification of E. coli possessing K88 and 987P pilus antigens.  相似文献   

11.
Seroagglutination (SAT), enzyme-linked immunosorbent assay (ELISA), and indirect fluorescent antibody staining tests (IFAT) were compared for reliability in the detection of pilus antigens K99, K88, and 987P of Escherichia coli. Test sensitivities were compared using mixtures of piliated bacteria of several strains diluted to a constant optical density with a nonpiliated strain. Relative sensitivities and specificities of the 3 tests were also compared using 55 E. coli strains that had previously been serotyped and characterized for pilus genes by DNA probe. Although specificity was not a serious problem with any of the tests, the SAT was relatively nonsensitive. The IFAT showed the greatest sensitivity of the 3 tests in detecting K88, K99, and 987P E. coli.  相似文献   

12.
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.  相似文献   

13.
Escherichia coli isolates from calves were investigated by multiplex PCR assays for the presence of genes encoding K99, F41, F17-related fimbriae, heat-stabile enterotoxin a (STa), intimin (eae) and Shiga toxins (stx1 and stx2). A total of 120 E. coli isolates, 75 isolated from diarrhoeic or septicemic calves and 45 from clinically healthy calves aged between 1 day and 2 months were tested. Each isolate was obtained from different calves in different herds. Among the isolates from diseased animals, 12 (16%) isolates from 1- to 7-day-old diarrhoeic calves were detected as enterotoxigenic E. coli which possessed K99, F41 and STa in combination; F17-related fimbriae genes were detected in 33 (44%) isolates and they were found in combination with K99 + F41 + STa in two isolates. Of 120 isolates, 16 carried eae, eight stx1 and five stx2 genes alone or in combination. None of the eae- or stx-positive strains was identified as O157:H7. However, results indicate that calves may be carrier of Shiga toxin-producing E. coli which have potential as a human pathogen. Antimicrobial susceptibility of 75 isolates from diseased calves was determined by agar disk diffusion method for 14 antimicrobial agents. In 77.3% of the isolates, multiresistance was detected. Higher resistance rates were detected for cephalothin (72%), tetracycline (69.3%), kanamycin (69.3%), ampicillin (65.3%), nalidixic acid (53.3%), trimethoprim-sulphamethoxazole (52%) and enrofloxacin (41.3%), respectively. No resistance was found for ceftiofur and cefoxitin.  相似文献   

14.
A total of 720 Escherichia coli strains isolated from diarrheic piglets on 756 swine farms were screened for the presence of the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR). Escherichia coli strains that carried EAST1 genes were also tested by PCR for the presence of 4 fimbriae (F4, F5, F6, F41), 2 heat-stable enterotoxins (STa and STb), and 1 heat-labile enterotoxin (LT) gene. One hundred sixty-four (22.7%) of the 720 E. coli isolates carried genes for EAST1. Of these 164 isolates, 62 (37.8%) carried EAST1 genes only, 11 (6.7%) carried genes for at least 1 of the fimbrial adhesins, 51 (31.1%) carried genes for at least 1 of the enterotoxins, and 40 (23.8%) carried genes for at least 1 of the fimbrial adhesins and enterotoxins. Forty-six percent of strains that carried EAST1 genes carried STa genes, and 16% of strains that carried EAST1 genes carried F4. The isolation rate of enterotoxigenic E. coli strains carrying genes for EAST1 gene was 63%. The 6 major genotypes observed in this study (in decreasing order) were EAST1+, EAST1+STa+, EAST1+STa+STb+, EAST1+STa+F5+, EAST1+STa+F4+, and EAST1+STb+F4+. EAST1 is widely prevalent among diarrheagenic strains of E. coli and may represent an important virulence determinant in the pathogenesis of enteric colibacillosis of preweaned pigs.  相似文献   

15.
Polymerase chain reaction for 4 fimbriae (F4, F5, F6, F41), 2 heat-stable enterotoxins (STa, STb), and 1 heat-labile enterotoxin (LT) were performed on 400 Escherichia coli isolates to determine their genotype prevalence among enterotoxigenic E. coli isolates from preweaned pigs with diarrhea in the Republic of Korea. A total of 200 of the 400 E. coli isolates were also selected for characterization of the O serogroup. Of these 200 isolates, serogroup could be determined in 139 (69.5%) but not in 61 isolates (30.5%). Isolates of serogroup O101 were the most common, followed in descending order by 08, 020, 0162, 0141, and 0149. Ninety-seven (24.3%) of the 400 E. coli isolates carried genes for at least 1 of the entertoxins or fimbrial adhesins. Of these 97 isolates, 27 carried genes for at least 1 of the fimbrial adhesins and entertoxins. Sixty-six percent of the isolates that carried fimbrial adhesin genes carried genes for at least 1 of the enterotoxins, and 71% of the isolates that carried enterotoxin genes carried genes for at least 1 of the fimbrial adhesins. Genes for the F6 fimbriae were detected in 6% of the E. coli isolates, and F4+, F41+, and F5+ genes were detected in 4.3%, 3.3%, and 2% of the isolates, respectively. Genes for STa, STb, and LT were detected in 10%, 8.5%, and 4.3% of the isolates, respectively. The 6 major genotypes observed in this study (in decreasing order) were F6+, STb+, F41+, STa+STb+, F6+STa+, and STa+.  相似文献   

16.
Three hundred and fifteen E. coli strains isolated from the intestine of piglets were examined for K-antigens 88 and 99, enterotoxin production and colicin resistance. Of these strains 308 belonged to one of 3 following different groups: Group 1: 0149, K88, producing heat-labile (LT) and heat-stable (ST) enterotoxins, group 2: 064, K99, producing ST, and group 3: variable O-antigens, no K-antigens or enterotoxin production.Almost 100 % of the E. coli strains were found to be resistant to colicins E1, E3, Ia, H and D+X. Resistance to colicins E2, B+M, V and K+X were found in 91.7 %, 43.8 %, 49.8 % and 62.2 % respectively.E. coli strains in group 1 were always (resistant to colicin E2, while about 87 % of the other strains were resistant to this colicin. E. coli strains in group 2 were more often resistant to colicin B+M, V and K+X (65 %, 94 %, 83 %) than strains in group 1 (37 %, 24 %, 64 %) and strains in group 3 (37 %, 52 %, 46 %).E. coli strains in group 2 showed a high degree of multiresistance, 45.1 % of the strains being resistant to all of the 9 colicins. About 10 % of the other strains were resistant to all of the 9 colicins.E. coli strains harbouring the enteropathogenicity factors K99 antigen and ST production, showed a higher degree of colicin resistance than both the E. coli strains with K88 antigen and ST and LT production, and the E. coli strains lacking enteropathogenicity factors.  相似文献   

17.
Eighty-eight of 1529 (5.7 per cent) Escherichia coli isolates from diarrhoeic and clinically normal calves in Scotland and northern England were found to possess the K99 pilus antigen (K99+). There was complete correlation between possession of K99 antigen, heat stable enterotoxin production and ability to dilate intestinal loops. The diagnosis of calf enterotoxigenic E coli infections may therefore be based on the detection of K99 antigen alone. Enterotoxigenic E coli was isolated from 23 of 306 (7.5 per cent) diarrhoeic calves from eight of 70 (11.4 per cent) farms and was not isolated from clinically normal calves. Infected calves were between one and three days old. A survey by an enzyme-linked immunosorbent assay found 3.0 per cent and 3.9 per cent of sera from calves and cows respectively to contain antibodies to K99 antigen. The prevalence of other enteropathogenic organisms in calf faeces is also discussed.  相似文献   

18.
Colony hybridizations with 4 enterotoxins (STaP, STaH, STb, and LT) and 1 adherence factor (K99) gene probes were done on Escherichia coli isolated from calves. Agreement between the K99 probing and a serologic assay to detect the K99 antigen was 99% for the identification of K99+ and K99- isolates. Ninety-five of the isolates (22%) hybridized with at least 1 enterotoxin gene probe (Ent+ isolates), and 82 (19%), with the K99 gene probe. The majority of Ent+ isolates (85%) reacted with probes for STaP and K99 genes. The STaP gene was present by itself in 4 of the Ent+ isolates (4%) and with the STb gene in 6 of the Ent+ isolates (6%). Five of the Ent+ isolates (5%) carried the STb and LT genes, and none (0%) of the isolates carried the STaH gene. All but 2 of the isolates with the K99 gene also had the STaP gene. Twenty-eight isolates shown to produce STa enterotoxin in previous studies failed to hybridize with any of the enterotoxin gene probes. These 28 isolates were also phenotypically negative when the tests for enterotoxin production were repeated. These isolates probably lost their genes for enterotoxin production during storage in the laboratory.  相似文献   

19.
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.  相似文献   

20.
Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0-11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes. These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41. Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones. The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones. Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources. Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.  相似文献   

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