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Necrotoxigenic escherichia coli (ntec) infection in farm animals: epidemiology and pathogenesis

Contract nr: FAIR-CT96-1335
Project nr: 1335
Project type: SC
Starting date: 01/01/1997
Duration: 36 months
Total cost: 899,100 EUR
EC Contribution: 734,900 EUR
Scientific Officer: Isabel MINGUEZ-TUDELA
Research topic: Animal health

Diarrhoeic and septicaemic colibacillosis of the neonates and young animals are the most common and devastating bacterial diseases in the farm animal industry. Most Escherichia coli strains are commensal inhabitants of the gastrointestinal tract, but some strains express virulence factors that enhance their ability to cause a wide variety of intestinal and extraintestinal diseases in calves, lambs, piglets, and human beings.

Besides enterotoxigenic, verotoxigenic, and enteropathogenic E. coli, there is evidence of the emergence of other types of pathogenic E. coli. One of the most recent group of strains whose importance was recognised are the so-called Necrotoxigenic E. coli. These are defined by the production of Cytotoxic Necrotising Factors (CNFs) 1 and 2, which induce necrosis in rabbit skin and multinucleation of eucaryotic cell cultures. The so-called necrotoxigenic E. coli (NTEC) must be recognised as a major developing problem on the basis of:

1) epidemiological data: NTEC1 are isolated from human beings, cattle, piglets, dogs, horses, and so on, with diarrhoea, septicaemia, urinary tract infections, and internal organs infections; NTEC2 are isolated from cattle and sheep with diarrhoea and septicaemia;

2) previous experimental infections in piglets and calves;

3) activity of CNFs and of NTEC on cell cultures;

4) association in clusters on the chromosome or on a plasmid of the cnf1 and cnf2 genes with genes coding for other putative virulence factors, especially adhesins of the P, S, F17, and Afa families.

The research goals of this proposal are:

1) the comparison of NTEC strains isolated in various European countries from cattle, humans and pigs, at the bacterial and molecular levels, by the study of their general, virulence-associated (including putative colonisation factors) and molecular properties (Part 1: Bacteriology and Epidemiology);
2) the definition of the contribution of the CNFs to the pathogenicity of NTEC strains at the molecular, cellular, and whole animal levels, by the modelling of the activity of the CNFs on different cell cultures and the definition of their contribution to the pathogenicity of NTEC strains in animals.

This objective was later extended to the study of the newly described Cytolethal Distending Toxins (CDT) (Part 2: Pathogenesis).

In the first part of the proposal, classical and virulence-associated properties of NTEC1 and NTEC2 strains will be studied, and their putative virulence factors and plasmids compared by classical and molecular epidemiology techniques. The putative colonisation factors will be precisely identified, characterised and compared at the molecular level with already described members of their respective families (P, S, F17, and Afa), with emphasis on putative new variant(s) of the Afa family. Finally, the diagnosis of the CNFs in routine diagnostic laboratories will be facilitated by the development of monoclonal antibody-based Elisa assays. The development of monoclonal antibody-based Elisa assays was extended to the adhesin antigens produced by NTEC.

In the second part, two different and complementary approaches will be followed to study the contribution of CNFs to the pathogenicity of NTEC strains:

1) in vivo models of infections in germ-free piglets with NTEC1 strains and in colostrum-deprived calves with NTEC2 strains;
2) in vitro models of interactions of NTEC1 and NTEC2 strains with epithelial cell cultures, either HeLa cells or polarised LLC-PK1 cells.
In both in vivo and in vitro models, the wild-type NTEC strains will be compared to their isogenic mutants in the cnf1 or cnf2 genes, obtained by allelic exchange. The production of mutants by allelic exchange was also extended to the cdt-3 gene coding for the Cytolethal Distending Toxins (CDT)-III in NTEC2, described during the project.

Current situation/results:
This FAIR research project ended on December 31, 1999. The final report is now completed. The summary of conclusions is described below.

In the conclusion of Part 1, it was neither possible to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. In addition, other adhesins are most probably produced by NTEC1 and/or NTEC2, as several of them are negative at the colony hybridisation assay with gene probes for P, S, F17 and Afa adhesins. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotising Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additional methods.

Application of Pulse Field Gel Electrophoresis might help in completion of NTEC1 strain comparison in particular cases of epidemiological investigation, keeping in mind that sometimes unrelated strains may present very similar, if not identical restriction profiles. NTEC1 strains have also been detected in relative high proportion from humans with urinary tract infection and NTEC2 strains from calves with enteritis in Central Europe. Their pathotypes are identical to those of NTEC strains isolated from other countries. The association of NTEC1, similar to human uropathogenic strains, and post-weaning diarrhoea in piglets, represents an original finding, because this type of NTEC1 is associated with invasive, rather than intestinal disease in animals.

Identification and typing of the P, S, F17 and Afa adhesins of NTEC1 and NTEC2 was achieved, and specific genetic diagnostic tools were developed (gene probes, PCRs) and can be used in a full typing scheme of any NTEC field isolate, after its identification as an NTEC. The new variant of the Afa family, Afa-VIII, appears to be widespread in animal E. coli and is also present in human E. coli. The existence of similar adhesin variants in animal and human E. coli does, however, not help in their full differentiation from an epidemiological point of view. If monoclonal antibody to CNF toxins were not produced, a specific F17b adhesin monoclonal was produced and used in a sandwich ELISA and in immunocytochemistry.

The studies also confirmed the linkage of cnf1 and prs gene clusters on a chromosomal pathogenicity island (PaiV) along with the hly genes coding for the haemolysin in animal and human NTEC1, and the linkage of cnf2 and f17 or afa-8 gene clusters on Vir plasmids belonging to the F incompatibility complex in bovine NTEC2. The chromosomal afa-8 gene cluster also appears to be located on a new undescribed pathothenicity island, tentatively named PAI I239KH89. The plasmid and chromosomal sequences flanking the afa-8 gene cluster differ, suggesting that the presence of afa-8 sequences in the chromosome did not originate from an integration of the Vir plasmid, nor that the plasmid localisation originates from a transposition event of the PAI I239KH89. Ten NTEC2 were f17 and afa-8 positive.

In the conclusion of Part 2, a new member of the family of Cytolethal Distending Toxins, CDT-III, was discovered and characterised. This CDT-III, like other CDTs, was able to trigger a mechanism of irreversible cell cycle arrest in G2 phase in active undifferentiated HeLa cells, that is initiated in S phase, through a different mechanism than DNA strand breaks, but possibly initiated during DNA replication. On differentiated quiescent cells (LLC-PK1), CNF2 and CDT-III would act synergistically: the production of CNF2, known to be mitogenic, is required for the activity of CDT-III on the cycle of quiescent confluent LLC-PK1 cells by pushing these quiescent cells into the S phase, where they then become receptive to the G2 blocking activity of CDT-III, which is dependent upon transit through a S phase. This synergy could be critical in vivo on fully differentiated epithelium and might impair intestinal barrier integrity. But the actual cellular receptors of the CNF and CDT toxins and the target of the CDT toxins still await identification.

NTEC1 and NTEC2 strains are able to reproduce clinical conditions in young piglets (septicaemia) and calves (diarrhoea and septicaemia). Low immune status of the animals, with hypo- or agammaglobulinaemia (colostrum deprivation or restriction) and absence of commensal intestinal flora (germ free or just born) appear to enhance the capability of the NTEC1 and NTEC2 to colonise their host and to cause disease. In these models NTEC strains can thus be considered as "opportunistic pathogens", waiting for favourable environmental circumstances, a consideration which in no way diminishes their virulent potential once these circumstances are present.

As to the role of the cytotoxins produced by NTEC strains in these models, it appears that the CNF2 toxin, but not the CDT-III toxin is important in the development of diarrhoea in calves infected with NTEC2 strains, whereas neither CNF1, nor CNF2, nor CDT-III plays any role in the development of septicaemia in piglets infected with NTEC1 and in calves infected with NTEC2 strains. These observations are of the utmost importance in the field of hygienic and vaccinal prophylaxis.

With respect to the objectives of the whole research project FAIR3 CT96-1335, the highlights are as follows:

  • the observation of direct, or indirect, role for CNF2 in the occurrence of diarrhoea in calves;
  • the description of a new Cytolethal Distending Toxin (CDT-III) variant produced by NTEC2;
  • the model of the interactions of CNF2 and CDT-III on the cell cycle of polarised cells;
  • the description of a new variant of the family of afimbrial adhesins (Afa-VIII), especially associated with E. coli of animal origin.

Jacques Georges MAINIL
Université de Liège
Sart Tilman
Bat. B43 A
B-4000 Liège
Tel.: +32 4 366 40 50
Fax: +32 4 366 41 22


  • Jean DE RYCKE
    Eric OSWALD
    Chemins des Capelles 23
    F-31076 Toulouse
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  • John Morris FAIRBROTHER
    Université de Montréal
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  • Chantal LE BOUGUENEC
    Institut Pasteur
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  • Hywel John BALL
    Queen's University of Belfast
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    UK-BT4 3SD Stormond - Belfast
    Tel.: +44 1232 52 57 05
    Fax: +44 1232 52 57 45

  • Bela NAGY
    Veterinary Medical Institute
    Hungarian Academy of Sciences
    Hungarian Str. 21
    H-1143 Budapest
    Tel.: +36 1 467 40 60
    Fax: +36 1 467 40 76
    E-mail: bnagy@novell.vmri.h

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