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Jacques Mainil - One of the best experts on this subject based on the ideXlab platform.
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Multiplex PCRs for Identification of Necrotoxigenic Escherichia Coli
Journal of clinical microbiology, 2003Co-Authors: Sigrid Van Bost, E. Jacquemin, Eric Oswald, Jacques MainilAbstract:Two multiplex PCRs were developed for the detection of Necrotoxigenic Escherichia Coli virulence genes. M1 contained the primers for the toxins and the aerobactin, and M2 contained the primers for the adhesins. They were validated by single PCRs performed with reference E. Coli strains and by multiplex PCRs with Necrotoxigenic E. Coli strains isolated from different animal species.
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Prevalence and identity of cdt-related sequences in Necrotoxigenic Escherichia Coli.
Veterinary microbiology, 2003Co-Authors: Jacques Mainil, E. Jacquemin, Eric OswaldAbstract:The cytolethal distending toxins (CDT) are responsible for the mitosis block at G2/M and the cycle arrest of cells in culture. Escherichia Coli isolated from humans and animals with intestinal and extra-intestinal diseases can be positive for the production of a CDT-like cytopathic effect or for the presence of cdt-related genes. The purpose of this study was to compare the prevalence and the identity of cdt-related sequences in Necrotoxigenic E. Coli (NTEC). A collection of 98 bovine type 2 NTEC (NTEC2) and 45 bovine, 20 canine, 3 feline, 65 human and 129 porcine type 1 NTEC (NTEC1) isolates was studied by colony hybridisation and PCR assays specific for the cdtB genes encoding the B sub-unit of the CDT-I, CDT-II, CDT-III and CDT-IV toxins produced by E. Coli. cdtB-III sequences were frequent amongst bovine NTEC2, since 83% of these isolates were positive by colony hybridisation and/or PCR, whereas cdtB-related sequences were rare amongst NTEC1, since only 2 bovine (4%), 3 canine (15%), 10 human (15%) and 13 porcine (10%) of these isolates were positive. The 28 probe-positive NTEC1 harboured cdtB-IV sequences (13 isolates), cdtB-I sequences (10 isolates), or still unidentified cdt-related sequences (5 isolates). After comparison with previously published and unpublished results of phenotypic assay on cell cultures, existence of other cdt-related sequences is suggested amongst NTEC1. The differences between NTEC1 and NTEC2 in their CDT profiles may have implication for the pathogenesis of those two classes of pathogenic E. Coli.
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Putative roles of the CNF2 and CDTIII toxins in experimental infections with Necrotoxigenic Escherichia Coli type 2 (NTEC2) strains in calves.
Microbes and infection, 2003Co-Authors: Sigrid Van Bost, Eric Oswald, Stefan Roels, Jacques MainilAbstract:Newborn colostrum-restricted calves were orally inoculated with an Escherichia Coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.
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Characteristics of Necrotoxigenic Escherichia Coli isolated from septicemic and diarrheic calves between 1958 and 1970.
Veterinary microbiology, 2001Co-Authors: Sigrid Van Bost, E. Jacquemin, Marie-hélène Bâbe, Jacques MainilAbstract:A total of 434 Escherichia Coli isolated from septicemic calves between 1958 and 1965 and 430 E. Coli isolated from diarrheic calves between 1967 and 1970 were studied by colony hybridisation and PCR assays for the presence of the cnf1- and the cnf2-like genes. They were also studied for the presence of genes coding for putative virulence factors associated with the CNF toxins including F17-, Pap- and Sfa-fimbrial adhesins and the recently described CDT-III toxin and AfaVIII-afimbrial adhesin. Thirty (7%) of the 434 septicemic strains were positive for CNF by colony hybridisation. Twenty-six were confirmed as Necrotoxigenic E. Coli type 2 (NTEC2) and four as NTEC1 by PCR. Thirty-five (8%) of the 430 diarrheic strains were positive for CNF by colony hybridisation. Five of them were studied by PCR and confirmed as NTEC1. The 26 septicemic NTEC2 strains and 20 of the 35 diarrheic NTEC including three of the five NTEC1 were positive for CDT-III. All adhesins studied were present in NTEC as well as in non-NTEC. NTEC1 were mainly Pap-, Sfa- and/or Afa8-positive, whereas NTEC2 were mainly F17- and/or Afa8-positive. This study shows that Necrotoxigenic E. Coli with their associated adhesins and toxins were present in calves as early as 1958, but their prevalence seems to have increased since that time.
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Necrotoxigenic Escherichia Coli type-2 invade and cause diarrhoea during experimental infection in colostrum-restricted newborn calves
Veterinary microbiology, 2001Co-Authors: Sigrid Van Bost, Stefan Roels, Jacques MainilAbstract:There exists experimental evidence that Necrotoxigenic Escherichia Coli (NTEC) strains producing the cytotoxic necrotising factor 1 cause intestinal and extra-intestinal disease in piglets. On the other hand, no experimental model has been developed with NTEC strains producing the cytotoxic necrotising factor 2. In all, 14 colostrum-restricted calves were orally challenged with two strains isolated from the faeces of a diarrheic calf (B20a) or from the heart blood of a septicaemic calf (1404). All calves had diarrhoea which lasted until euthanasia in eight of them. In those calves, diarrhoea was correlated with the faecal excretion of the challenge strains. At necropsy, vascular congestion of the intestinal mucosa, hypertrophy of the mesenteric lymph nodes (MLN) and some congestion of the lungs were observed. Bacteriology confirmed the colonisation of the intestine by the challenge strains which were also recovered from the heart blood, the lungs and/or the liver. Histological sections confirmed enteroColitis, lymphadenitis and limited bronchopneumonia. In the intestinal tissue sections, bacteria testing positive in an in situ DNA hybridisation assay with a CNF2 probe were observed. Those results were confirmed by immunohistochemistry with a polyclonal anti-O78 and a monoclonal anti-F17b antisera. Three of the five control calves receiving either saline or a CNF(-), F17a strain (25KH09) had no clinical signs or lesions. The other two presented a profuse liquid diarrhoea but those calves were positive for the presence of K99(+) E. Coli. In this model, both NTEC2 strains were thus, able to colonise the intestine, to cause long-lasting diarrhoea and to invade the blood stream with localisation in various internal organs in colostrum-restricted conventional newborn calves.
E. Jacquemin - One of the best experts on this subject based on the ideXlab platform.
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Multiplex PCRs for Identification of Necrotoxigenic Escherichia Coli
Journal of clinical microbiology, 2003Co-Authors: Sigrid Van Bost, E. Jacquemin, Eric Oswald, Jacques MainilAbstract:Two multiplex PCRs were developed for the detection of Necrotoxigenic Escherichia Coli virulence genes. M1 contained the primers for the toxins and the aerobactin, and M2 contained the primers for the adhesins. They were validated by single PCRs performed with reference E. Coli strains and by multiplex PCRs with Necrotoxigenic E. Coli strains isolated from different animal species.
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Prevalence and identity of cdt-related sequences in Necrotoxigenic Escherichia Coli.
Veterinary microbiology, 2003Co-Authors: Jacques Mainil, E. Jacquemin, Eric OswaldAbstract:The cytolethal distending toxins (CDT) are responsible for the mitosis block at G2/M and the cycle arrest of cells in culture. Escherichia Coli isolated from humans and animals with intestinal and extra-intestinal diseases can be positive for the production of a CDT-like cytopathic effect or for the presence of cdt-related genes. The purpose of this study was to compare the prevalence and the identity of cdt-related sequences in Necrotoxigenic E. Coli (NTEC). A collection of 98 bovine type 2 NTEC (NTEC2) and 45 bovine, 20 canine, 3 feline, 65 human and 129 porcine type 1 NTEC (NTEC1) isolates was studied by colony hybridisation and PCR assays specific for the cdtB genes encoding the B sub-unit of the CDT-I, CDT-II, CDT-III and CDT-IV toxins produced by E. Coli. cdtB-III sequences were frequent amongst bovine NTEC2, since 83% of these isolates were positive by colony hybridisation and/or PCR, whereas cdtB-related sequences were rare amongst NTEC1, since only 2 bovine (4%), 3 canine (15%), 10 human (15%) and 13 porcine (10%) of these isolates were positive. The 28 probe-positive NTEC1 harboured cdtB-IV sequences (13 isolates), cdtB-I sequences (10 isolates), or still unidentified cdt-related sequences (5 isolates). After comparison with previously published and unpublished results of phenotypic assay on cell cultures, existence of other cdt-related sequences is suggested amongst NTEC1. The differences between NTEC1 and NTEC2 in their CDT profiles may have implication for the pathogenesis of those two classes of pathogenic E. Coli.
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Characteristics of Necrotoxigenic Escherichia Coli isolated from septicemic and diarrheic calves between 1958 and 1970.
Veterinary microbiology, 2001Co-Authors: Sigrid Van Bost, E. Jacquemin, Marie-hélène Bâbe, Jacques MainilAbstract:A total of 434 Escherichia Coli isolated from septicemic calves between 1958 and 1965 and 430 E. Coli isolated from diarrheic calves between 1967 and 1970 were studied by colony hybridisation and PCR assays for the presence of the cnf1- and the cnf2-like genes. They were also studied for the presence of genes coding for putative virulence factors associated with the CNF toxins including F17-, Pap- and Sfa-fimbrial adhesins and the recently described CDT-III toxin and AfaVIII-afimbrial adhesin. Thirty (7%) of the 434 septicemic strains were positive for CNF by colony hybridisation. Twenty-six were confirmed as Necrotoxigenic E. Coli type 2 (NTEC2) and four as NTEC1 by PCR. Thirty-five (8%) of the 430 diarrheic strains were positive for CNF by colony hybridisation. Five of them were studied by PCR and confirmed as NTEC1. The 26 septicemic NTEC2 strains and 20 of the 35 diarrheic NTEC including three of the five NTEC1 were positive for CDT-III. All adhesins studied were present in NTEC as well as in non-NTEC. NTEC1 were mainly Pap-, Sfa- and/or Afa8-positive, whereas NTEC2 were mainly F17- and/or Afa8-positive. This study shows that Necrotoxigenic E. Coli with their associated adhesins and toxins were present in calves as early as 1958, but their prevalence seems to have increased since that time.
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The afa-related gene cluster in Necrotoxigenic and other Escherichia Coli from animals belongs to the afa-8 variant.
Veterinary microbiology, 2000Co-Authors: J. Gérardin, E. Jacquemin, C Le Bouguénec, Lila Lalioui, Jacques MainilAbstract:Six hundred and nine Necrotoxigenic Escherichia Coli type 1 and 2 (NTEC1 and NTEC2) and non-NTEC isolated in Western and Southern Europe, North Africa and Canada from diseased calves, pigs, humans, poultry, and 55 isolated from asymptomatic calves were studied for the identification of afa-related sequences to the recently described afa-7 and afa-8 gene cluster variants from two bovine Escherichia Coli (Lalioui et al., 1999). Colony hybridization and PCR assays for the afaD-7, afaE-7, afaD-8 and afaE-8 identified the afa-related sequences to the afa-8 gene cluster in most (67/79; 85%) of the E. Coli positive with the Afa-f family probe and in 14 additional strains negative with the Afa-f probe. No E. Coli was positive for the afa-7 gene cluster. The existence of afa-8 positive strains was thus confirmed among bovine E. Coli and for the first time among porcine, poultry and human E. Coli. Sequencing of the afaE-8 amplicon of nine strains from the different host species showed a high degree of conservation (>95% at the DNA level; >92% at the amino-acid level). The afa-8 gene cluster was more frequent in E. Coli from diseased calves (18%) than from piglets (12%), humans (6%) and poultry (5%). Bovine NTEC2 (26%) were more frequently positive than NTEC 1 (20%) and non-NTEC (11%). E. Coli isolated from asymptomatic calves were rarely positive: one NTEC2 (3%) and no non-NTEC. The afa-8 gene cluster was located on the Vir plasmid in 11/23 NTEC2, but no plasmid localization was detected in NTEC1 or non-NTEC.
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Identification of the F17 fimbrial subunit- and adhesin-encoding (f17A and f17G) gene variants in Necrotoxigenic Escherichia Coli from cattle, pigs and humans.
Veterinary microbiology, 2000Co-Authors: Jacques Mainil, J. Gérardin, E. JacqueminAbstract:Putative colonization factors of the F17 family of fimbrial adhesins have been identified in Necrotoxigenic Escherichia Coli Type 1 and Type 2 (NTEC1 and NTEC2) from calves, pigs, and humans. The f17A and f17G gene variants, coding respectively for the major subunit and for the adhesin of the F17 fimbriae, were typed in 70 E. Coli carrying f17-related sequences (15 NTEC1, 51 NTEC2, and four non-NTEC) by colony hybridisation with gene probes derived from the different f17A gene variants (a, b, c, and d) and by PCRs specific for each f17A and f17G (I and II) gene variants. Typing of f17A genes was not possible by colony hybridisation, as most 70 E. Coli were positive with more than one gene probe. On the other hand, the PCRs allowed the typing of the f17A gene in 37 E. Coli and of the f17G gene in all 70 E. Coli. The f17Ab gene variant was detected in 13 NTEC2; the f17Ac, in all 15 NTEC1, six NTEC2 and two non-NTEC; and the f17Ad, in one non-NTEC. Seven additional NTEC2 were positive with the PCRs for two variants: f17Ab and f17Ac in three of them; f17Ac and f17Ad in four of them. Either these seven NTEC2 harbour two variants or the variant present can be detected by two PCRs. The remaining 25 NTEC2 and one non-NTEC tested negative with the PCRs for the four f17A gene variants, suggesting the existence of other variant(s). In contrast, all 70 E. Coli were positive with the PCR for the f17GII gene variant and none with the PCR for the f17GI gene variant. The f17-related sequences were present on the CNF2/Vir plasmids in 27 out of the 46 NTEC2 from which plasmid DNA could be extracted: all but one of those positive for the f17Ab gene variant and various proportions of those positive for other variants. In contrast, no plasmid carried f17-related sequences in NTEC1 and non-NTEC.
Miguel Blanco - One of the best experts on this subject based on the ideXlab platform.
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Necrotoxigenic Escherichia Coli from sheep and goats produce a new type of cytotoxic necrotizing factor (CNF3) associated with the eae and ehxA genes.
International microbiology : the official journal of the Spanish Society for Microbiology, 2007Co-Authors: José A. Orden, Miguel Blanco, Jesús E. Blanco, Azucena Mora, Jorge Blanco, Gustavo Domínguez-bernal, Susana Martínez-pulgarín, Ricardo De La FuenteAbstract:Fecal samples from sheep and goats were screened by tissue-culture assays and PCR for the presence of Necrotoxigenic Escherichia Coli (NTEC) producing cytotoxic necrotizing factors (CNFs). Of the 18 NTEC strains assayed, four were positive for the cnf1 gene while 14 strains were negative for the cnf1 and cnf2 genes. All of the NTEC strains had the eae gene and most of them also carried the ehxA gene. Moreover, all the cnf1- cnf2- NTEC strains were negative for several virulence markers associated with CNF1+ or CNF2+ strains. The cnf gene present in one of these strains was sequenced and analysis of the gene product revealed a new type of CNF, which was named CNF3 (and the coding gene cnf3). Oligonucleotide primers were designed to PCR-amplify a fragment of cnf3. The results showed that all strains examined in this study, except one cnf1+strain, were cnf3+. The association of cnf3 with eae and ehxA suggests that cnf3+ NTEC strains might be pathogenic for humans.
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Distribution and characterization of faecal Necrotoxigenic Escherichia Coli CNF1+ and CNF2+ isolated from healthy cows and calves.
Veterinary microbiology, 1998Co-Authors: Miguel Blanco, Jesús E. Blanco, Azucena Mora, Jorge BlancoAbstract:Faecal swabs obtained from a random sample of 268 cows and 90 calves on 19 Lugo (northwestern Spain) farms were examined for Necrotoxigenic Escherichia Coli (NTEC) producing the cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2). We found NTEC CNF1+ and CNF2+ on 11% and 95% of the farms, respectively. NTEC producing CNF2 were significantly more frequently isolated from calves (58%) than from cows (17%) (P < 0.001). The proportion of animals colonized with CNF2+ strains on each farm ranged from 0% to 60%. NTEC strains producing CNF2 isolated from healthy cattle belonged to 27 O serogroups; however, 64% were of one of 12 serogroups (O2, O8, O8–O75, O14, O15, O55, O86, O88, O115, O121, O147, and O168). Furthermore, the serogroups determined in CNF2+ strains isolated from cows (O2, O8, and O14) were different from those found in NTEC producing CNF2 isolated from calves (O8–O75, O15, O55, O86, O88, O115 and O147).
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Prevalence and characteristics of Necrotoxigenic Escherichia Coli CNF1+ and CNF2+ in healthy cattle
Research in microbiology, 1998Co-Authors: Miguel Blanco, Jesús E. Blanco, Azucena Mora, Jorge BlancoAbstract:From February to July of 1994, 328 faecal samples from 32 herds were collected and examined for Necrotoxigenic Escherichia Coli (NTEC). Strains producing the cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2) were found on 4 and 63% of the farms, respectively. The proportion of animals infected within each herd varied from 0 to 38%. NTEC producing CNF2 were significantly more frequently isolated from calves (24%; 17 of 71) than from cows (4%; 11 of 257) (χ2corr. 25.088; P < 0.001). Although the bovine CNF2+ strains belonged to 16 different serogroups, 5 (O15, O77, O88, O142 and O153) accounted for 44% of strains. This study confirmed that healthy cattle are a reservoir of NTEC producing CNF2, and revealed that CNF2+ strains are more frequently carried by calves than by adult cows.
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Polymerase chain reaction for detection of Escherichia Coli strains producing cytotoxic necrotizing factor type 1 and type 2 (CNF1 and CNF2)
Journal of Microbiological Methods, 1996Co-Authors: Miguel Blanco, Jesús E. Blanco, Juan Blanco, María Pilar Alonso, Carlos Balsalobre, M. Mouriño, Cristina Madrid, Antonio JuárezAbstract:Abstract Cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2) are dermonecrotic protein toxins produced by human and animal clinical isolates of Escherichia Coli . In this study, two pairs of oligonucleotide primers were designed to amplify fragments of the genes for CNF1 and CNF2 by the polymerase chain reaction (PCR). We also described a further primer pair which amplify fragments of both CNF1 and CNF2 genes. Amplification products of the expected size (543-bp for CNF1, 543-bp for CNF2 and 533-bp for CNFs) were detected in all 240 Necrotoxigenic Escherichia Coli (NTEC) strains tested. In contrast, no amplification products were seen when the PCR reaction was performed with DNA obtained from 69 non-NTEC bacterial strains (including 13 enterotoxigenic, 11 verotoxigenic and 45 non-toxigenic strains). Furthermore, the toxin genotypes determined by PCR corresponded to the phenotypic results of HeLa cell assay. Therefore, the specificity and sensitivity for the three pairs of oligonucleotide primers designed in this study was 100%. The PCR protocol describe here permits rapid and accurate detection of CNF1 and CNF2 genes in Escherichia Coli strains isolated from clinical material and contaminated foods.
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Serotypes of bovine Escherichia Coli producing cytotoxic necrotizing factor type 2 (CNF2).
Veterinary microbiology, 1994Co-Authors: Miguel Blanco, Jesús E. Blanco, Jorge Blanco, Anjo J. Verbruggen, W. H. JansenAbstract:The serotypes of 101 faecal bovine Necrotoxigenic Escherichia Coli (NTEC) producing the cytotoxic necrotizing factor type 2 (CNF2) were determined. Although, NTEC producing CNF2 belonged to 48 different O:K:H serotypes, only eleven of them accounted for 54% of strains. The most common serotypes in order of frequency were: O123:K-:H16, O3:K-:H21, O88:K-:H8, O15:K14:H21, O1:K-:H12, O1:K1:H46, O2:K1:H5, O55:H21, O88:K?:H25, O117:K?:H21 and O123:K-:H-. The serotypes of CNF2 NTEC were different from those found in NTEC producing CNF1 and in enterotoxigenic, verotoxigenic, enteropathogenic, enteroinvasive and enteroadherent E. Coli strains that cause infections in humans and animals.
Eric Oswald - One of the best experts on this subject based on the ideXlab platform.
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Divergent evolution of the repFII replicon of incF plasmids carrying cytotoxic necrotizing factor cnf2, cytolethal distending toxin cdtIII, and f17Ae fimbrial variant genes in type 2 Necrotoxigenic Escherichia Coli isolates from calves
Applied and Environmental Microbiology, 2016Co-Authors: Morgan Bihannic, Eric Oswald, Marisa Haenni, Jean-yves MadecAbstract:Among the pathovars of Escherichia Coli in cattle, Necrotoxigenic E. Coli (NTEC) is defined by the production of cytotoxic necrotizing factors (CNFs). In particular, type 2 NTEC (NTEC2) strains are frequent in diarrheic and septicemic calves and usually coproduce CNF type 2 (CNF2), cytolethal distending toxin type III (CDTIII), and fimbrial adhesins of the F17 family, whose genetic determinants have frequently been reported on the same Vir-like plasmid. In this study, we investigated the genetic environment of the cnf2, f17Ae, and cdtIII genes in a collection of fecal E. Coli isolates recovered from 484 French and 58 Iranian calves. In particular, we highlighted the spread of cnf2, f17Ae, and cdtIII on similar 150-kb IncF plasmids harboring the newly assigned repFII replicon allele F74 in NTEC2 isolates. Interestingly, this 150-kb IncF plasmid differed from the 140-kb IncF plasmid harboring the newly assigned repFII replicon allele F75 and carrying cnf2 alone. These results suggest two divergent lineages of cnf2-carrying IncF plasmids depending on the presence of the f17Ae and cdtIII genes. This partition was observed in E. Coli strains of unrelated backgrounds, suggesting two different evolutionary paths of cnf2-carrying IncF plasmids rather than divergent evolutions of NTEC2 clones. The driving forces for such divergent evolutions are not known, and further studies are required to clarify the selection of plasmid subtypes spreading virulence determinants in E. Coli, in particular, plasmids of the IncF family.
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Multiplex PCRs for Identification of Necrotoxigenic Escherichia Coli
Journal of clinical microbiology, 2003Co-Authors: Sigrid Van Bost, E. Jacquemin, Eric Oswald, Jacques MainilAbstract:Two multiplex PCRs were developed for the detection of Necrotoxigenic Escherichia Coli virulence genes. M1 contained the primers for the toxins and the aerobactin, and M2 contained the primers for the adhesins. They were validated by single PCRs performed with reference E. Coli strains and by multiplex PCRs with Necrotoxigenic E. Coli strains isolated from different animal species.
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Prevalence and identity of cdt-related sequences in Necrotoxigenic Escherichia Coli.
Veterinary microbiology, 2003Co-Authors: Jacques Mainil, E. Jacquemin, Eric OswaldAbstract:The cytolethal distending toxins (CDT) are responsible for the mitosis block at G2/M and the cycle arrest of cells in culture. Escherichia Coli isolated from humans and animals with intestinal and extra-intestinal diseases can be positive for the production of a CDT-like cytopathic effect or for the presence of cdt-related genes. The purpose of this study was to compare the prevalence and the identity of cdt-related sequences in Necrotoxigenic E. Coli (NTEC). A collection of 98 bovine type 2 NTEC (NTEC2) and 45 bovine, 20 canine, 3 feline, 65 human and 129 porcine type 1 NTEC (NTEC1) isolates was studied by colony hybridisation and PCR assays specific for the cdtB genes encoding the B sub-unit of the CDT-I, CDT-II, CDT-III and CDT-IV toxins produced by E. Coli. cdtB-III sequences were frequent amongst bovine NTEC2, since 83% of these isolates were positive by colony hybridisation and/or PCR, whereas cdtB-related sequences were rare amongst NTEC1, since only 2 bovine (4%), 3 canine (15%), 10 human (15%) and 13 porcine (10%) of these isolates were positive. The 28 probe-positive NTEC1 harboured cdtB-IV sequences (13 isolates), cdtB-I sequences (10 isolates), or still unidentified cdt-related sequences (5 isolates). After comparison with previously published and unpublished results of phenotypic assay on cell cultures, existence of other cdt-related sequences is suggested amongst NTEC1. The differences between NTEC1 and NTEC2 in their CDT profiles may have implication for the pathogenesis of those two classes of pathogenic E. Coli.
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Putative roles of the CNF2 and CDTIII toxins in experimental infections with Necrotoxigenic Escherichia Coli type 2 (NTEC2) strains in calves.
Microbes and infection, 2003Co-Authors: Sigrid Van Bost, Eric Oswald, Stefan Roels, Jacques MainilAbstract:Newborn colostrum-restricted calves were orally inoculated with an Escherichia Coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.
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Presence of pap-, sfa-, and afa-related sequences in Necrotoxigenic Escherichia Coli isolates from cattle: evidence for new variants of the AFA family.
Canadian Journal of Veterinary Research, 1997Co-Authors: J Mainil, E. Jacquemin, Frédéric Herault, Eric OswaldAbstract:Necrotoxigenic Escherichia Coli (NTEC) are associated with intestinal and extraintestinal diseases in animals and human beings and produce Cytotoxic Necrotizing Factor 1 (CNF1) or 2 (CNF2). Fourty-three NTEC1, 42 NTEC2, and 32 CNF-negative isolates from cattle were tested by colony DNA hybridization, by plasmid DNA hybridization and by PCR assays for the presence of DNA sequences homologous to the operons coding for fimbrial (PAP/PRS, SFA/FIC, and F17) and afimbrial (AFA/Dr) adhesins of extraintestinal E. Coli. Most NTEC1 isolates hybridized with the PAP probes and either the SFA probe (37%) or the AFA probes (49%). Most NTEC2 isolates, in contrast, hybridized with the F17 probe (45%), the AFA probes (19%), or the F17 and AFA probes (22%). A probe-positive plasmid was identified in each of the 19 NTEC2 isolates studied. They all hybridized with the CNF2 toxin probe (Vir plasmids) and most of them with the F17 (6 plasmids) or AFA (7 plasmids) probes. PCR amplification was obtained with 6 of the 11 NTEC isolates tested for the papGII/prsG genes; with all 5 NTEC isolates tested for the sfa and related operons; but with none of the 18 NTEC isolates tested for the afa and related operons. pap-, sfa-, and afa-related sequences are thus present in NTEC isolates from cattle in addition to f17-related operons and may code for adhesins corresponding to specific colonization factors. f17- and afa-related sequences can be located on the Vir plasmids along with the cnf2 gene. Existence of new variants of the AFA/Dr family is evident from the negative results of this family-specific PCR assay.
Sigrid Van Bost - One of the best experts on this subject based on the ideXlab platform.
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Multiplex PCRs for Identification of Necrotoxigenic Escherichia Coli
Journal of clinical microbiology, 2003Co-Authors: Sigrid Van Bost, E. Jacquemin, Eric Oswald, Jacques MainilAbstract:Two multiplex PCRs were developed for the detection of Necrotoxigenic Escherichia Coli virulence genes. M1 contained the primers for the toxins and the aerobactin, and M2 contained the primers for the adhesins. They were validated by single PCRs performed with reference E. Coli strains and by multiplex PCRs with Necrotoxigenic E. Coli strains isolated from different animal species.
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Putative roles of the CNF2 and CDTIII toxins in experimental infections with Necrotoxigenic Escherichia Coli type 2 (NTEC2) strains in calves.
Microbes and infection, 2003Co-Authors: Sigrid Van Bost, Eric Oswald, Stefan Roels, Jacques MainilAbstract:Newborn colostrum-restricted calves were orally inoculated with an Escherichia Coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.
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Characteristics of Necrotoxigenic Escherichia Coli isolated from septicemic and diarrheic calves between 1958 and 1970.
Veterinary microbiology, 2001Co-Authors: Sigrid Van Bost, E. Jacquemin, Marie-hélène Bâbe, Jacques MainilAbstract:A total of 434 Escherichia Coli isolated from septicemic calves between 1958 and 1965 and 430 E. Coli isolated from diarrheic calves between 1967 and 1970 were studied by colony hybridisation and PCR assays for the presence of the cnf1- and the cnf2-like genes. They were also studied for the presence of genes coding for putative virulence factors associated with the CNF toxins including F17-, Pap- and Sfa-fimbrial adhesins and the recently described CDT-III toxin and AfaVIII-afimbrial adhesin. Thirty (7%) of the 434 septicemic strains were positive for CNF by colony hybridisation. Twenty-six were confirmed as Necrotoxigenic E. Coli type 2 (NTEC2) and four as NTEC1 by PCR. Thirty-five (8%) of the 430 diarrheic strains were positive for CNF by colony hybridisation. Five of them were studied by PCR and confirmed as NTEC1. The 26 septicemic NTEC2 strains and 20 of the 35 diarrheic NTEC including three of the five NTEC1 were positive for CDT-III. All adhesins studied were present in NTEC as well as in non-NTEC. NTEC1 were mainly Pap-, Sfa- and/or Afa8-positive, whereas NTEC2 were mainly F17- and/or Afa8-positive. This study shows that Necrotoxigenic E. Coli with their associated adhesins and toxins were present in calves as early as 1958, but their prevalence seems to have increased since that time.
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Necrotoxigenic Escherichia Coli type-2 invade and cause diarrhoea during experimental infection in colostrum-restricted newborn calves
Veterinary microbiology, 2001Co-Authors: Sigrid Van Bost, Stefan Roels, Jacques MainilAbstract:There exists experimental evidence that Necrotoxigenic Escherichia Coli (NTEC) strains producing the cytotoxic necrotising factor 1 cause intestinal and extra-intestinal disease in piglets. On the other hand, no experimental model has been developed with NTEC strains producing the cytotoxic necrotising factor 2. In all, 14 colostrum-restricted calves were orally challenged with two strains isolated from the faeces of a diarrheic calf (B20a) or from the heart blood of a septicaemic calf (1404). All calves had diarrhoea which lasted until euthanasia in eight of them. In those calves, diarrhoea was correlated with the faecal excretion of the challenge strains. At necropsy, vascular congestion of the intestinal mucosa, hypertrophy of the mesenteric lymph nodes (MLN) and some congestion of the lungs were observed. Bacteriology confirmed the colonisation of the intestine by the challenge strains which were also recovered from the heart blood, the lungs and/or the liver. Histological sections confirmed enteroColitis, lymphadenitis and limited bronchopneumonia. In the intestinal tissue sections, bacteria testing positive in an in situ DNA hybridisation assay with a CNF2 probe were observed. Those results were confirmed by immunohistochemistry with a polyclonal anti-O78 and a monoclonal anti-F17b antisera. Three of the five control calves receiving either saline or a CNF(-), F17a strain (25KH09) had no clinical signs or lesions. The other two presented a profuse liquid diarrhoea but those calves were positive for the presence of K99(+) E. Coli. In this model, both NTEC2 strains were thus, able to colonise the intestine, to cause long-lasting diarrhoea and to invade the blood stream with localisation in various internal organs in colostrum-restricted conventional newborn calves.
