Rapid identification of virulence genes in enterotoxigenic Escherichia coli isolates associated with diarrhoea in Queensland piggeries

OBJECTIVE: To identify virulence genes in enterotoxigenic E coli (ETEC) isolates associated with diarrhoea in neonatal, 1 to 3 week-old and weaned pigs in southeast Queensland. DESIGN: Multiplex PCR and serotyping were applied to E coli isolates obtained over a 5-year period (1998-2002) from cases diagnosed at Toowoomba Veterinary Laboratory. PROCEDURE: A total of 126 isolates from 25 different Queensland piggeries were tested for haemolytic activity on 5% sheep blood agar and by multiplex PCR for the presence of five commonly recognised fimbrial (F4, F5, F6, F41 and F18) and three enterotoxin genes (STa, STb, LT). A subset of 62 representative isolates were serotyped by slide agglutination. For comparative purposes, multiplex PCR was also performed on the DNA of 31 ETEC isolates from 9 serotypes originating from piggeries in southern New South Wales. RESULTS: A total of 113 (89.7%) of the isolates from Queensland possessed ETEC virulence genes, including 14 of 15 isolates from neonatal pigs (93.3%), 18 of 23 isolates from 1 to 3 week old pigs (78.3%) and 81 of 88 isolates from weaned pigs (92.1%). F4:STa:STb:LT (serotype O149) was the most prevalent pathotype in neonatal and 1-3 week old pigs and F4:STa:STb:LT (serotype O149) and F18:STa:STb:LT (serotype O141) were most prevalent in weaned pigs. In comparison, isolates obtained from neonatal pigs from New South Wales belonged to a more diverse range of pathotypes and serotypes. CONCLUSION: Multiplex PCR was a rapid and specific method for detecting the presence of ETEC virulence genes in porcine E coli isolates. For isolates obtained from cases of suspected colibacillosis in Queensland, growth of a heavy pure culture of haemolytic E coli was a sensitive prognostic indicator of the presence of ETEC virulence genes in the isolate. ETEC pathotypes and serotypes remained stable in Queensland piggeries over the five-year study period and appear to have changed little over the last three decades.     

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.     

Isolation and association of Escherichia coli AIDA-I/STb, rather than EAST1 pathotype, with diarrhea in piglets and antibiotic sensitivity of isolates.

To identify emerging Escherichia coli that have the potential to cause diarrhea in pigs, the prevalence of E. coli pathotypes was determined among 170 and 120 isolates from diarrheic and nondiarrheic piglets, respectively. The isolates were tested for F4, F5, F6, F18, and F41 fimbriae, for E. coli attaching and effacing (EAE), porcine attaching and effacing-associated (Paa), and adhesin involved in diffuse adherence (AIDA-I) factors, for LT, STa, STb, and enteroaggregative heat-stable (EAST1) enterotoxins, and for Shiga toxins (Stxl, Stx2, and Stx2e), using DNA hybridization and polymerase chain reaction. All isolates were O-serotyped and tested for antibiotic resistance against 10 drugs. Seventeen different pathotypes, accounting for 40.0% of the isolates, were recovered from diarrheic piglets. The main pathotypes included EAST1 (13.5%), F4/LT/STb/EAST1 (6.5%), AIDA-I/STb/EAST1 (4.1%), F5/STa (2.9%), EAE/EAST1 (2.9%), and AIDA-I/F18 (2.3%). Only 3 pathotypes, EAE (11.7%), EAST1 (10.8%), and EAE/EAST1 (3.3%), were recovered from nondiarrheic piglets. Paa factor was detected in 8.8% and 7.5% of isolates from diarrheic and nondiarrheic piglets, respectively, and always was associated with other virulence determinants. Overall, 22.9% of isolates from diarrheic piglets appeared to be enteropathogens: enterotoxigenic E. coli (11.7%), enteropathogenic E. coli (3.5%), and E. coli isolates (3.0%) for which none of the above adherence factors was detected. Pathotypes AIDA-I/STb/EAST1 and AIDA-I/STb were isolated only from diarrheic piglets and accounted for 4.7% of isolates. Strains of these pathotypes induced diarrhea when inoculated into newborn colostrum-deprived pigs, in contrast to an isolate positive only for EAST1, which did not induce diarrhea. Antibiotic sensitivity test showed that isolates of the AIDA-I/STb/EAST1 and AIDA-I/STb pathotypes were the only strains sensitive to enrofloxacin, gentamicin, neomycin, and trimethoprim-sulfamethoxazole. This study showed that at least 20.5% of isolates from diarrheic piglets appeared to be associated with AIDA-I/STb pathotype and that EAST1 pathotype is probably not an important marker for diarrhea in piglets.     

Replicon typing of F18 fimbriae encoding plasmids of enterotoxigenic and verotoxigenic Escherichia coli strains from porcine postweaning diarrhoea and oedema disease

The presence of fimbrial adhesin F18 is frequently found in enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) strains responsible for diarrhoea and oedema disease of weaned pigs. The F18 adhesin occurs in two antigenic variants: F18ab is characteristic of VTEC while F18ac is more typical for ETEC. F18 encoding plasmids of 17 phenotypically characterized porcine E. coli isolates (10 ETEC, 6 VTEC and 1 ETEC/VTEC) were tested with a DNA probe for F18 fimbrial adhesin and with replicon probes for the RepFIa, RepFIb and for the RepFIc family of basic replicons. In all the cases, the F18 probe hybridized to only one plasmid band of size higher than 42MDa. All F18 plasmids were determined to be unireplicon plasmids belonging to the RepFIc replicon family of the F incompatibility complex. There was no difference between F18ac plasmids of ETEC and F18ab plasmids of VTEC strains in terms of replicon type or subtype. However, the size of F18ab plasmids of the VTEC strains varied between 42 and 98MDa, in contrast to F18ac plasmids of ETEC strains (constantly approximately 98MDa).     

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.     

Distribution of virulence genes in Escherichia coli strains isolated from diarrhoeic piglets in the Slovak Republic

Ninety-two Escherichia coli isolates from 14 to 28-day-old piglets that died because of diarrhoea were examined for genes for fimbriae (F4, F5, F6, F18 and F41), enterotoxins (STa, STb and LT), verotoxin (VT2e or Stx2e) and enteroaggregative heat-stable enterotoxin 1 (EAST1) by polymerase chain reaction. Twenty-two strains (24%) carried a gene for F4, whereas genes for F18, F6 and F5 + F41 were detected in 10.8, 3.3 and 1.1% of strains respectively. Genes for STb, LT, STa and Stx2e were detected in 40.2, 26.1, 14.1 and 1.1% of strains respectively. The astA gene was detected in 49 (53.3%) isolates, 35 of which also carried genes for enterotoxins and/or fimbriae. The major genotypes reached at (in decreasing order of prevalence) were F4/STb/LT/EAST1, F18/STa/STb/EAST1, STb/EAST1, F6/STa/STb/EAST1 and F18/STb/EAST1.     

Isolation of Escherichia coli from piglets in South Korea with diarrhea and characteristics of the virulence genes

Escherichia coli was isolated from the feces of 122 piglets with diarrhea on 55 farms in Korea. The virulence genes of each isolate were characterized by polymerase chain reaction (PCR). Of the 562 isolates, 191 carried 1 or more of the virulence genes tested for in this study. Of the 191 isolates, 114 (60%) carried 1 or more of the genes for enterotoxigenic E. coli (ETEC) fimbriae F4, F5, F6, F18, and F41 and ETEC toxins LT, STa, and STb, 57 (30%) carried 1 or more of the genes for the Shiga-toxin-producing E. coli (STEC) toxins Stx1, Stx2, and Stx2e, and 21% and 37% carried the gene for enteropathogenic E. coli intimin and for enteroaggregative E. coli toxin, respectively. Collectively, our results indicate that other pathotypes of E. coli as well as ETEC can be strongly associated with diarrhea in piglets. In addition, detection of the genes for Stx1 and Stx2 indicates that pigs are reservoirs of human pathogenic STEC.     

Prevalence of enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> virulence genes from scouring piglets in Zimbabwe

World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe.We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened in toxin positive isolates. Toxin positive strains lacking tested fimbriae genes were characterized using transmission electron microscopy, agglutination and agglutination inhibition tests. Approximately 32% of the 1,984 isolates tested positive for STa, STb, LT or Stx-2e genes. Of these, approximately 81% had F4, F5, F6, F18 or F41 fimbriae genes. The remaining toxin positive strains lacked tested fimbriae genes and appeared to either express F1-like fimbriae, or lacked fimbriae. The data constitute an important framework for implementation of prevention measures, such as using relevant fimbriae-based vaccines against ETEC induced diarrhea or VTEC-induced edema.     

Development of multiplex polymerase chain reaction assays for detecting enterotoxigenic Escherichia coli and their application to field isolates from piglets with diarrhea.

Fimbriae and enterotoxins are major virulence factors associated with enterotoxigenic Escherichia coli (ETEC). In this study, 3 sets of multiplex polymerase chain reaction (mPCR) assays targeting fimbriae, enterotoxins, and other adherence factors were developed for detecting ETEC. A total number of 188 E. coli field isolates were examined, and percentages of E. coli strains carrying each virulence factors were as follows: F4 (7.45%), F5 (29.79%), F6 (6.38%), F18 (15.43%), F41 (3.72%), STa (10.11%), STb (20.74%), LT (9.57%), Stx2e (2.13%), EAST1 (42.02%), F1 (67.55%), AIDA-I (2.66%), and pAA (7.45%). Of the 188 E. coli field isolates examined, 25.53% were found to be pathogenic ETEC, having both fimbriae and enterotoxins. However, the ratio increased to 44.68% when the presence of other adhesins was considered as criteria for virulence. Among the adherence factors, F1 was found to be the most prevalent. AIDA-I and pAA were also found with similar ratio as compared with other virulence factors. In addition, virulence patterns carrying these alternate adhesive genes with enterotoxins were detected with significant ratio. Therefore, it is desirable that alternate adhesins be considered as markers for diagnosis of ETEC.     

Prevalence of genotypes for fimbriae and enterotoxins and of O serogroups in Escherichia coli isolated from diarrheic piglets in Korea.

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+.     

Comparative prevalence of four enterotoxin genes among Escherichia coli isolated from swine

Presence of Escherichia coli enterotoxin genes LT (heat-labile enterotoxin), STaP (heat-stable enterotoxin a, porcine genotype), STaH (heat-stable enterotoxin a, human genotype), and STb (heat-stable enterotoxin b) among 874 swine isolates of E coli was determined, using DNA probes and the DNA colony hybridization technique. Of the 874 isolates evaluated, 45% hybridized with at least one of the enterotoxin gene probes and were designated as enterotoxigenic E coli (ETEC). Eighty-five percent of the ETEC were from pigs with enteric colibacillosis. The remaining 15% were from pigs with edema disease or various other diseases, and from healthy swine. Seventy-four percent of the ETEC hybridized with the STb probe, 52% with STaP, and 31% with LT; ETEC did not hybridize with the STaH probe. Most of the ETEC hybridized with more than one enterotoxin gene probe. Isolates that hybridized with the LT probe also hybridized with STb. The most prevalent gene combination was LT-STb. However, 35% of the ETEC from neonatal (less than or equal to 1 week old) swine with enteric colibacillosis were of the STaP-only genotype, and 33% of the ETEC from older swine with enteric colibacillosis were of the STb-only genotype.     

Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene and its relationship with fimbrial and enterotoxin genes in E. coli isolated from diarrheic piglets.

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.     

Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia

Two hundred and fifty Escherichia coli isolates from diarrhoeic and healthy piglets were serotyped and tested for the presence of virulence genes for fimbriae, intimin, heat-labile (LT) and heat-stable (STa and STb) enterotoxins, Stx toxins, and enteroaggregative heat-stable 1 (EAST1) enterotoxin by polymerase chain reaction (PCR). Although 220 isolates from diarrhoeic piglets belonged to 43 O serogroups and 77 O:H serotypes, 60% were of one of the 10 serogroups O2, O8, O15, O54, O84, O101, O141, O147, O149 and O157, and 60% belonged to only 10 serotypes (O8:H-, O54:H-, O84:H7, O101:H-, O141:H-, O141:H4, O147:H-, O149:H10, O163:H-, and ONT:H-). PCR showed that 79% of 220 isolates carried genes for at least one of the virulence factors tested. The gene encoding for EAST1 was the most prevalent (65%) followed by those encoding for STb (49%), LT (42%), STa (13%), and Stx2e (4%). Eighty-three (38%) of the 220 E. coli isolates carried the gene for F4 (K88), whereas genes for F18, F5 (K99), F41, F6 (P987), F17, and intimin (eae) were detected in 9%, 3%, 3%, 3%, 1%, and 3%, respectively. Seropathotype O149:H10:F4:LT/STb/EAST1 (70 isolates) was the most common, representing 32% of isolates. Pulsed-field gel electrophoresis (PFGE) analysis with XbaI of 15 O149:H10 representative isolates from diarrhoeic piglets distinguished 14 types. The 15 isolates exhibited a wide variability of distinct restriction patterns though all belonged to the same serotype (O149:H10), and all but one showed identical virulence determinants (F4, LT, STb, and EAST1). Among 30 isolates from healthy piglets only two virulence genes were detected: EAST1 (26%) and eae (17%). In total, 12 isolates were positives for the eae gene: five isolates had intimin beta1, four possessed intimin theta and three showed intimin type xiB. This is believed to be the first study describing the presence of intimin type xiB in E. coli of porcine origin.     

Hybridization of bovine Escherichia coli isolates with gene probes for four enterotoxins (STaP, STaH, STb, LT) and one adhesion factor (K99)

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.     

Virulence factors in Escherichia coli isolated from the blood of bacteremic neonatal calves

Twenty-five Escherichia coli isolates from the blood of critically ill bacteremic calves sampled in two separate studies on a calf-rearing farm housing over 15,000 calves, in the San Joaquin Valley, California were studied.Isolates were characterized for O serogroups and for pathotypes as determined by the presence of specific virulence factors including heat-labile enterotoxin (LT), heat-stable enterotoxins a and b (STa, STb), verotoxins 1 and 2 (VT1, VT2), cytotoxic necrotizing factor (CNF), aerobactin, intimin Eae and P, F17 and CS31A fimbrial adhesins, and resistance to bactericidal effects of serum.These isolates constituted a heterogeneous group. However, isolates were mostly aerobactin positive and often resistant to the bactericidal effects of serum. Isolates of pathotypes O78 (n=6), O119:CS31a (n=3), and P positive but O non-typeable (n=3) were associated with a high mortality rate. The remaining isolates belonged to diverse pathotypes, often possessing the adhesins P, F17, CS31A and Eae but belonging to O serogroups other than O78 and O119, and were less frequently associated with mortality.Although no virulence factor common to all isolates was identified, the capacity to use iron by the presence of aerobactin which is important to the capture of iron was a predominant factor. Moreover, certain pathotypes appear to be associated with primary colisepticemia whereas other pathotypes may cause a bacteremia without necessarily leading to septicemia.     

Genotypic prevalence of the fimbrial adhesins (F4, F5, F6, F41 and F18) and toxins (LT, STa, STb and STx2e) in Escherichia coli isolated from postweaning pigs with diarrhoea or oedema disease in Korea

A PCR was used to determine the genotypic prevalence of five fimbrial adhesins (F4, F5, F6, F41 and F18), two heat-stable enterotoxins (STa and STb), the heat-labile enterotoxin (LT), and the shiga toxin 2e (Stx2e) in 230 isolates of Escherichia coli from postweaning pigs with diarrhoea or oedema disease. Ninety-four (40.9 per cent) of the isolates carried genes for at least one of the fimbrial adhesins or toxins. Genes for the F18 fimbrial adhesin were detected in 18.3 per cent, and genes for F4, F6, F5 and F41 were detected in 10.0 per cent, 4.3 per cent, 1.7 per cent and 0.8 per cent of the isolates, respectively. Genes for STa, STb and LT were detected in 25.7 per cent, 15.2 per cent and 8.7 per cent of the isolates, respectively. Genes for Stx2e were detected in 36 (15.6 per cent) of the isolates, and among them 24 also contained the gene for F18ab and four also contained the gene for F18ac.     

Prevalence of serogroups and virulence genes in Escherichia coli associated with postweaning diarrhoea and edema disease in pigs and a comparison of diagnostic approaches

Identification of Escherichia coli causing porcine postweaning diarrhoea (PWD) or edema disease (ED) requires knowledge regarding the prevalent pathotypes within a given region. This study was undertaken to determine the present distribution of serogroups, hemolytic activity and virulence factor gene profiles among porcine pathogenic E. coli isolates in Denmark and to compare detection of these characteristics as diagnostic approaches. Five hundred and sixty-three E. coli were serogrouped using E. coli O-antisera and investigated for hemolytic activity. Of these, 219 isolates were further characterized using a 5'-nuclease PCR assay detecting genes for adhesion factors, enterotoxins and verocytotoxin 2e (VT2e). Forty-two different serogroups were found. The most prevalent serogroup was O149 accounting for 49.9% of all isolates, followed by O138 (14.9%), O139 (6.9%), O141 (4.1%) and O8 (3.7%). Hemolytic activity was detected in 87.7% of all isolates. Virulence factor genes detected were F4 (44.7%), F18 (39.3%), intimin (1.4%), F6 (0.9%), STb (77.6%), EAST1 (65.8%), LT (61.6%), STa (26.5%) and VT2e (16.4%). Six pathotypes accounted for 65.7% of all isolates investigated. Using possession of virulence factor genes as reference, O-serogrouping employing a selection of antisera representing common pig pathogenic serogroups and detection of hemolysis were evaluated as epidemiological markers for pathogenicity. Both criteria were associated with pathogenicity (P<0.001, for both), however, both methods also resulted in false classifications regarding pathogenicity for 11.9 and 13.2% of isolates, respectively. Detection of adhesion factor genes F4, F18 and intimin is suggested as an operational alternative when diagnosing PWD and ED.     

PCR detection of virulence factor genes in Escherichia coli isolates from weaned piglets with edema disease and/or diarrhea in China

Fimbriae, toxins and pathogenicity islands (PAIs) are main virulence factors of the pathogenic Escherichia coli strains. To investigate into their prevalence in clinical E. coli isolates associated with porcine postweaning diarrhea (PWD) and/or pig edema disease (ED), 240 isolates were obtained from diseased piglets (140 from PWD, 76 from ED and 24 from ED/PWD) and submitted to PCR detection for genes coding for fimbriae, enterotoxins, shiga toxins, intimin and high-molecular-weight protein 2 (HMWP2). Among the 240 isolates detected, detection rates of the genes for F18, F4, intimin, HMWP2, Stx2e, LTa, STa and STb were 26.25%, 3.75%, 28.33%, 16.67%, 35%, 10.83%, 14.58% and 9.17%, respectively, and 67.92% of the isolates could be assigned into 20 different virulence factor patterns. Further more, F18ab+ STEC are the prevalent pathogens of ED, and F18+ and/or intimin+ STEC/ETEC are the dominant pathogens of ED/PWD, while F18ab+, F4+ and/or intimin+ ETEC and HPI+ and/or LEE+ E. coli are more frequently associated with PWD.     

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.     

Genotypic prevalence of the adhesin involved in diffuse adherence in Escherichia coli isolates in pre-weaned pigs with diarrhoea in Korea

A total of 1002 Escherichia coli strains isolated from pre-weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat-stable (STa, STb) and heat-labile (LT) enterotoxin, enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty-three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre-weaned pigs with diarrhoea.