The transfer of immunity from the mother to the neonate through milk was demonstrated in the mouse in 1892 by P. Ehrlich. The intestinal absorption of the colostrum and its importance for the calf survival have been long studied and nobody doubts about the colostral origin of the acquired passive immunity of the calf, though very little is known about the mechanism of immunoglobulin absorption.

The general purpose of this work was to study the mechanism of intestinal immunoglobulin absorption and to identify factors influencing this absorption. Antibody absorption was evidenced by blood analysis. The passage through the intestinal mucosa was studied at the cellular level using gammaglobulins marked with fluorochromes, a quite new technique.

Some preliminary assays on young rats proved that conjugation with fluorochromes does not interfere with the intestinal absorption in this species. In the neonatal calf, after injection under global anesthesia of conjugated proteins in ligated intestinal loops, we also proved that neither marking nor anesthesia interfered with absorption in this species. We could thus pursuit our work with small amounts of marked proteins.

The study comprised the following points:

• duration of intestinal permeability to the gammaglobulins;
• localisation of intestinal absorption of antibodies;
• way of this absorption;
• influence of other proteins on the absorption of immunoglobulins;
• duration of the absorption;
• some aspects of the proteinuria that follows the absorption of immunoglobulins;
• absorption of the IgM immunoglobulins and of some other proteins.

The experiments were performed on newborn calves or on ligated intestinal loops of newborn calves.

The results obtained, in three experiments, confirm that the intestinal absorption of colostrum globulins occurs mainly during the first and, to a lesser extent, during the second day of life. Traces of absorbed globulins are detectable in certain calves aged from 52 to 53 hours. We could not find any absorption in calves of 60 hours of age and more. The results were always identical, whether the globulins were labelled (fluorescein isothiocyanate or Rhodamine B 200) or not, given "per os" or injected directly into the intestinal loops. The presence of large amounts of amniotic fluid in the abomasum may retard the passage of colostrum to the intestine and hence reduce the absorption of the globulins.

The absorption of the proteins, by the small intestine of the newborn calf, occurs mainly in the jejunal portion. No absorption takes place neither in the anterior part of the duodenum nor in the terminal part of the ileum.

In the newborn calf, the colostrum gammaglobulins, which are absorbed by pinocytosis, mainly in the jejunum, reach the circulation exclusively through the lymphatic channels.

In the newborn calf and during the period of intestinal permeability to proteins: i) the presence of proteins of colostrum in the blood does not interfere with the intestinal absorption of subsequently ingested colostral immunoglobulins; ii) the intestinal epithelial cells which have absorbed a former protein remain capable to absorb the gammaglobulins administrated at a later period.

The epithelial cells of the small intestine of the newborn calf are capable of absorbing the soluble molecules of protein by pinocytosis. This capacity decreases in the next generations of these cells. The aptitude for pinocytosis of protein is probably lost after one or two replacements of the intestinal epithelium. This gives a probable explanation for the cessation of the intestinal absorption of colostrum globulins in the newborn calf.

After intestinal absorption of labelled colostral immunoglobulins by neonatal calves, some (or some of their fragments) are excreted in urines while retaining their labelling. Different protein fractions are present in the urine: the most important reacts with an anti-gamma globulin immunserum; a second one, though reacting with the same immunserum is less motile by electrophoresis; a third fraction corresponds to the serumalbumin. After glomerular filtration, some proteins (or some of their fragments) are reabsorbed by pinocytosis by the epithelial cells of the proximal convoluted tubule only, never by epithelial cells of the other parts of the nephron.

Gammaglobulins are absorbed through the lymphatics, bovine serumalbumine follows both lymphatic and venous pathway, while the beta-lactoglobulin and the alpha-lactalbumin are absorbed mainly through the venous channels. In our study, we cannot decide if it is only their molecular weights which determine their pathway or other special characteristics of each protein.

Schoenaers F., Kaeckenbeeck A. and El-Nageh M. Prophylaxie de la colibacillose du veau par vaccination de la mère. Ann Méd Vét, 1967, 111: 3-15.

El-Nageh M. Période de perméabilité de l'intestin du veau nouveau-né aux gammaglobulines du colostrum. Ann Méd Vét, 1967, 111: 370-379.

El-Nageh M. Siège de l'absorption intestinale des gammaglobulines du colostrum chez le veau nouveau-né. Ann Méd Vét, 1967, 111: 380-383.

El-Nageh M. Voies d'absorption des gammaglobulines du colostrum au niveau de l'intestin grêle du veau nouveau-né. Ann Méd Vét, 1967, 111: 384-390.

Kaeckenbeeck A., El-Nageh M. and Schoenaers F. Effets d'une première administration de protéines sur la résorption intestinale ultérieure des globulines, chez le veau nouveau-né. Ann Méd Vét, 1967, 111: 391-399.

El-Nageh M. Relation entre l'arrêt de la résorption intestinale des anticorps et le renouvellement de l'épithélium intestinal. Ann Méd Vét, 1967, 111: 400-405.

El-Nageh M. A propos de la protéinurie du veau nouveau-né. Quelques observations sur l'excrétion urinaire et la réabsorption tubulaire des globulines marquées. Ann Méd Vét, 1968, 112: 479-485.

El-Nageh M. Absorption intestinale des protéines chez le veau nouveau-né. Différence dans les voies empruntées par certaines protéines. Ann Méd Vét, 1968, 112: 699-703.

Necrotoxigenic Escherichia coli (NTEC) are defined on the basis of the production of a cytotoxin causing occurence of giant multinucleated cells (CHO, Vero, HeLa, HEp-II) in culture and necrosis after intradermal injection in rabbits or intrafoodpad in mice and therefore named Cytotoxic Necrotizing Factor (CNF). Two CNF toxins differing by their biological, antigenic and genetic properties have been described, CNF1 and CNF2. NTEC are associated with various digestive, urinary and systemic infections in mammals: NTEC1 in humans, domestic and some wild mammals; NTEC2 almost exclusively in domestic and wild ruminants.

The purpose of the work reported here was to study the general and putative virulent, classical and molecular properties of a collection of bovine, canine, feline, human and porcine NTEC1 strains and of bovine NTEC2 strains, to: i) define the identity card of NTEC1 and NTEC2 strains in an attempt to better understand their putative virulence factors and to improve their diagnosis; and ii) compare and differentiate the NTEC1 strains isolated from humans and animals in the possible case of a public health problem.

The typing assays were either phenotypic for the production of aerobactin (Aer), of colicin (Col), and of haemolysin (Hlyalpha, Hlybeta and entero-Hly), for the resistance to the bactericidal activity of the serum (RS), for the serotyping, and for the biotyping, or genetic for the CNF1 and CNF2 toxins, for the Cytolethal Distending Toxins (CDT-I, -II, -III, -IV), for the fimbrial adhesins of the P (Pap/Prs), S (Sfa/F1C), and F17 (a, b, c, d) families, and for the afimbrial adhesins of the Afa family (Afa, Nfa, Dr, F1845, M). Colony hybridization was used as a first screening assay to recognise the presence of homologous DNA sequences within the strains. Polymerase Chain Reaction (PCR) was subsequently used to type the variant(s) of the gene(s) detected, coding for the different CNF (cnf1, cnf2) and CDT (cdtB-I, cdtB-II, cdtB-III, cdtB-IV) toxins or for the different P (papGI, papGII, papGIII), S (sfa, foc), F17 (f17Aa, f17Ab, f17Ac, f17Ad, f17GI, f17GII) and Afa (afa8-D, afa8-E) fimbrial and afimbrial adhesins.

During a first study, genetic assays were developed and standardized. DNA probes were derived from the gene coding for the CNF2 toxin after its cloning: a first probe fragment detected the NTEC2 strains (pEOSW03 fragment), while a second probe fragment could detect, and differentiate, both NTEC1 and NTEC2 strains (pEOSW01 fragment). In parallel, probes to detect genes coding for the CDT toxins, for fimbrial adhesins of the P, S and F17 families and for afimbrial adhesins of the Afa family were derived and tested on a collection of bovine NTEC1 and NTEC2 strains, to obtain standardized experimental conditions for the future studies.

During a second study all phenotypic and genetic assays previously designed were applied on collections of NTEC strains of bovine (45 NTEC1 and 98 NTEC2 strains), canine (99 NTEC1 strains), feline (33 strains), human (65 strains), and porcine (129 strains) origin. The results obtained can be summarized as follows. The majority of NTEC1 strains from cats, dogs, humans and pigs were positive with probes for the P (80-91%) and/or S (74-86%) fimbrial adhesins. F17 and Afa probe-positive NTEC1 strains were much rarer (0-12%). Bovine NTEC1 strains gave similar results with the P (75% of probe-positive strains) and F17 (7% of probe-positive strains) probes, but were distinguished by lower frequency of S probe-positive (42%) and higher frequency of Afa probe-positive (20%) strains. The other frequently positive properties were production of Hlyalpha/beta (81-100%) and RS (82-96%). Aer was produced by variable proportions of bovine, human and porcine NTEC1 strains (478%), and by only 4% of canine strains. The other genes or properties were too rarely detected (CDT <4-15%>, entero-Hly <0-9%>, Col <22-34%> or too variable (serotypes, biotypes), to be considered as useful in the typing of NTEC1 strains. The bovine NTEC2 strains were mainly positive for the F17 fimbrial (52%) and/or Afa afimbrial (24%) adhesins, and for a CDT toxin (83%). In contrast, strains positive for the P and/or S fimbrial adhesins were anecdotic (6%). To the other assays, the bovine NTEC2 strains gave results comparable to those obtained with NTEC1 strains, with one main difference, i.e. the lower number of strains producing a Hlyalpha/beta (37%).

During a third study the probe-positive strains were tested with the appropriate PCR assays to identify the variant(s) of the gene detected by colony hydridization. Most of the results obtained with NTEC1 and NTEC2 strains were various for the CDT toxins and for the antigenic and adhesin variant(s) of the P, S, and F17 fimbrial adhesins. The main two exceptions to this variability were the bovine NTEC2 strains which harboured the cdtB-III gene coding for the CDT-III toxin, and all animal and human NTEC1 and NTEC2 strains which harboured the afa8-D and afa8-E genes coding for the Afa-VIII afimbrial adhesin.

At the end a general identity profile could be defined for the NTEC1 strains: CFN1+ P and/or S+ Hlyalpha/beta+ RS+ which corresponds to 73% of the 45 bovine, 91% of the 23 canine, 88% of the 65 human and 75% of the 129 porcine strains studied. This profile could even be more precisely defined for the canine NTEC1 strains: CNF1+ PapGIII and/or S+ Hlyalpha/beta+ RS+ biotype9+. In contrast the most frequent identity profile of the bovine NTEC2 strains: CNF2+ CDT-III+ F17 and/or Afa-VIII+ RS+, grouped only half (40%) of the 98 strains studied.

According to these results, no assay can replace individually the search for the production of CNF1 or CNF2 toxin, or for their encoding genes, in the diagnosis of NTEC strains. So test result associations were also examined, more especially the associations between Hlyalpha/beta and fimbriae P from the one hand, or sorbose fermentation (biotypes 1, 2, 3, 4, 9, 10, 11 and 12) from the other hand.

The first association was indeed very frequent amongst bovine, canine, human and porcine NTEC1 strains (64-81%). The second association was even more frequent amongst 78-100% of bovine, canine and human NTEC1, but not amongst porcine NTEC1 strains (50%). Neither association was frequent amongst NTEC2 strains. These results do not however allow to propose these assays for the diagnosis of NTEC1 strains, since several non-NTEC strains can be positive for these associations of properties.

As a conclusion, our research studies confirmed and completed the identity profiles of bovine, canine, feline, human and porcine NTEC1 and bovine NTEC2 strains, and of some of their potential virulence properties according the host and the bacteria: P, S, F17 and/or Afa adhesins as colonisation factors; CNF and CDT toxins to cross the intestinal mucosal barrier and/or to cause toxic effects on the host tissues; production of an aerobactin and of an alpha or beta hemolysin and resistance to the bactericidal activity of the serum for survival within the host blood stream and internal tissues and organs.

On the other hand, the results did not allow the development of a new methodology in the diagnosis of NTEC1 and/or NTEC2 strains, nor bring new arguments in the debate over a potential role of animal NTEC1 strains as zoonotic agents. Indeed animal and human strains were positive for the adhesins belonging to the same families (P, S, F17 and/or Afa) and carrying the same antigenic and adhesin variants (PapGII or PapGIII; Sfa or F1C; F17Ac ande F17GII; Afa-VIII).

Oswald E., Pohl P., Jacquemin E., Lintermans P., Van Muylem K., O'Brien A.D. and Mainil J. Specific DNA probes to detect Escherichia coli strains producing cytotoxic necrotising factor type 1 or type 2. J Med Microbiol, 1994, 40: 428-434.

Mainil J., Jacquemin E., Hérault F. and Oswald E. Presence of pap-, sfa-, and afa-related sequences in necrotoxigenic Escherichia coli isolates from cattle: evidence for new variants of the AFA family. Can J Vet Res, 1997, 61: 193-199.

Mainil J., Jacquemin E., Pohl P., Fairbrother J.M., Ansuini A., Le Bouguénec C., Ball H.J., De Rycke J. and Oswald E. Comparison of necrotoxigenic Escherichia coli isolated from farm animals and from humans. Vet Microbiol, 1999, 70: 123-135.

Mainil J., Jacquemin E. and Oswald E. Prevalence and identity of cdt-related sequences in necrotoxigenic Escherichia coli. Vet Microbiol, 2003, 94: 159-165.

Mainil J., Gérardin J. and Jacquemin E. Identification of the F17 fimbrial subunit- and adhesin-encoding (f17A and f17G) gene variants in necrotoxigenic Escherichia coli from cattle, pigs and humans. Vet Microbiol, 2000, 73: 327-335.

Gérardin J., Lalioui L., Jacquemin E., Le Bouguénec C. and Mainil J. The afa-related gene cluster in necrotoxigenic and other Escherichia coli from animals belonging to the afa-8 variant. Vet Microbiol, 2000, 76: 175-184.

Mainil J., Bex S., Jacquemin E. and Kaeckenbeeck A. Les souches pathogènes d'Escherichia coli chez les chiens et chats: I) Détection des souches entérotoxinogènes (ETEC), entéropathogènes (EPEC), vérotoxinogènes (VTEC), entérohémorragiques (EHEC) et nécrotoxinogènes (NTEC). Ann Méd Vét, 1998, 142: 39-46.

Mainil J., Wilbaux M., Jacquemin E., Oswald E., Imberechts H. and Van Bost S. Les souches pathogènes d'Escherichia coli chez les chiens et chats: III) Données bactériologiques et cliniques sur les souches nécrotoxinogènes et sur celles positives pour des adhésines. Ann Méd Vét, 2001, 145: 343-354.

One thousand one hundred eighty-nine Escherichia coli isolated from young calves (less than one month-old) with clinical signs or lesions of enteric and/or systemic colibacillosis have been studied by DNA-DNA colony hybridization with radioactively labelled gene probes derived from the genes coding for four enterotoxins (STaP, STaH, STb and LT) and one adhesin (K99) produced by enterotoxigenic strains of Escherichia coli (ETEC). These E. coli isolates belonged to three collections : two collections of the bacteriology laboratory of the veterinary Faculty of the University of Liège (one from 1967-1970 and one from 1979 to 1986) and one American collection from 1961-1982 (USDA, ARS, National Animal Disease Centre, Ames, Iowa ; Dr H.W. Moon).

Two hundred and twenty E. coli isolates tested positive with at least one of the five gene probes. The agreement between the hybridization of the STaP probe and the suckling mouse assay was 100%, and the agreement between the hybridization of the K99 probe and the agglutination assay with an anti-K99 immunserum was >99%. The major pathotype was STaP+K99+ (90% of the probe positive isolates). The other 21 isolates were STaP+, K99+, STaP+STb+ or STb+LT+.

Since the gene coding for the different enterotoxins and for the K99 adhesin of ETEC have been located on plasmids, plasmid DNA was extracted from 18 STaP+K99+ bovine ETEC and three STaP+K99+ porcine ETEC. The plasmids were separated according to their molecular weight by electrophoresis in agarose gels that were dehydrated and directly hybridized with the radioactively labelled gene probes. In all isolates the STaP and K99 encoding genes were located on the same high molecular weight plasmid band (between 70 and 120 Mdaltons). In three isolates the STaP probe also hybridized with a second plasmid band. Attempts to transfer these plasmids by conjugation or mobilization were unsuccessful.

To more precisely identify and compare these STaP+K99+ plasmids their basic replicons were identified with gene probes derived from the basic replicons of plasmids belonging to the following incompatibility groups : FIA, FIB, RepFIIA family, HI1, HI2, HII, L/M, N, P, T, U, W, X and Y. At first 116 ETEC isolates and 116 non-ETEC isolates were tested with the replicon-derived gene probes by colony hybridization. The FIA, FIB and RepFIIA probes were hybridized by a majority of ETEC isolates (between 85% and 100%) and by many non-ETEC isolates (between 30% and 70%). The other replicon probes were hybridized by less than one third of the isolates or by none at all. Seventeen of the STaP+K99+ plasmids hybridized with the three replicon probes (FIA, FIB and RepFIIA family) and most probably carry three basic replicons. The other four STaP+K99+ plasmids hybridized with only two replicon probes: the RepFIIA family probe and either the FIA or the FIB replicon probes, and therefore most probably carry only two basic replicons.

As a conclusion, the majority of bovine ETEC harbour the genes coding for the STaP enterotoxin and K99 adhesin that are located on a single plasmid. These STaP+K99+ plasmids are multireplicons, carrying one FIA and/or one FIB basic replicon, along with one basic replicon of the RepFII family

Mainil J., Bex F., Couturier M., Kaeckenbeeck A. Caractérisation d'Escherichia coli entérotoxiques d'origine bovine par hybridation ADN-ADN sur colonies. Ann Rech Vet, 1984, 15: 139-144.

Mainil J., Bex F., Couturier M., Kaeckenbeeck A. Diagnostic de la colibacillose entérotoxique du veau au moyen de deux sondes ST: application sur le contenu intestinal. Ann Méd Vét, 1984, 128: 467-470.

Mainil J., Bex F., Couturier M., Kaeckenbeeck A. Hybridization on bovine enterotoxigenic Escherichia coli with two heat-stable enterotoxin gene probes. Am J Vet Res, 1985, 46: 2582-2584.

Mainil J.G., Moseley S.L., Schneider R.A., Sutch K., Casey T.A., Moon H.W. Hybridization of bovine Escherichia coli isolates with gene probes for four enterotoxins (STaP, STaH, STb, LT) and one adhesion factor (K99). Am J Vet Res, 1986, 47: 1145-1148.

Mainil J., Kaeckenbeeck A. Les souches d'Escherichia coli entérotoxingènes du veau produisent un seul type de toxine active chez le souriceau: STaP. Ann Méd Vét, 1986, 130: 531-534.

Mainil J., Bex F., Dreze P., Couturier M., Kaeckenbeeck A. Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolated from cattle. Infect Immun, 1992, 60: 3376-3380.

Since the beginning of the century, a few Clostridium perfringens typing methods have been described; toxintyping by seroneutralisation of mice lethality is the most widely used. Today molecular genetics allows to characterise much easier this species.

On the one hand, genes coding for most of the toxins were searched by colony DNA-DNA hybridisation (alpha-, beta-, epsilon-, iota-, theta- and mu-toxins, enterotoxin and sialidase) or by polymerase chain reaction (alpha-, beta-, epsilon- and iota-toxins and enterotoxin). In order to obtain nucleotid sequence of the epsilon-toxin gene, a 5144 bp DNA fragment of C. perfringens was cloned from the extrachromosomal DNA fraction of type-D strain NCTC 2062 by means of an oligonucleotide DNA probe and a stepwise cloning procedure, and the complete sequence realized. One thousand one hundred and twenty-three wild-type strains were studied with the gene probes. For example, the 768 strains isolated from bovine enterotoxaemia cases in Belgium possess always alpha-toxin and sialidase genes, often the theta- and mu-toxin genes, rarely the enterotoxin gene and never the beta-, epsilon- and iota-toxin genes.

On the other hand, a C. perfringens epidemiological marker was defined by HindIII restriction fragment polymorphism study after hybridisation with five probes, one specific for 16S rRNA gene and 4 specific for sialidase, alpha-, theta- and mu-toxin genes. This method allowed to confirm, on 166 isolates, the lack of genetic homogeneity of the different toxintypes and the virulence genes mobility in this bacterial species. The isolates from cattle were genetically very different between several animals but also in one individual animal. Thus, many different toxinotype A clones could cause the bovine enterotoxaemia in Belgium.

Finally, studies were performed about an insertion sequence, named IS1151, that we have identified on the epsilon-toxin gene fragment. This sequence (1696 bp) possesses two 21 bp stretches, one at each extremity, which constitute a perfect inverted repeat. Strong homology was found with IS231 of Bacillus thurigiensis. Like IS231 in Bacillus thurigiensis, IS1151 may be involved in virulence gene transfer in C. perfringens. Indeed, a colony hybridization study performed with an IS1151 probe internal to the ORF sequence revealed homologous sequences in all type B and type D isolates but also in some non-epsilon-toxin-producing strains: in all 6 type C strains (beta-toxin-producing), in one type E strain (iota-toxin-producing), in 18 out of 48 enterotoxin-producing type A strains, but only in a very few non-enterotoxigenic type A strains (352 of 2594). Moreover, some type C strains having lost the capacity to produce beta-toxin failed to hybridize with the IS1151 probe. The IS1151-like element was located near the epsilon-toxin structural gene in all 40 type B, and type D isolates studied by Southern blotting and near the beta-toxin, iota-toxin, or enterotoxin structural genes in other toxinotypes. The IS1151-like elements associated with the different toxin genes were cloned and sequenced in order to determine the phylogeny of these structures.

Daube G. Clostridium perfringens et pathologies digestives. Ann Méd Vét, 1992, 136: 5-30.

Daube G., Simon P. and Kaeckenbeeck A. IS1151, an IS-like element of Clostridium perfringens. Nucleic Acids Researchs, 1993, 21: 352.

Daube G., Simon P., Limbourg B., Renier K. and Kaeckenbeeck A. Typage moléculaire de Clostridium perfringens. Ann Méd Vét, 1994, 138: 183-191.

Daube G., China B., Simon P., Hvala K. and Mainil J. Typing of Clostridium perfringens by in vitro amplification of toxin genes. J Appl Bacteriol, 1994, 77: 650-655.

Cornillot E., Saint-Joanis B., Daube G., Canard B., Granum P.-E and Cole S.T. Variable location of the enterotoxin Gene (cpe) in Clostridium perfringens. Mol Microbiol, 1995, 15: 639-645.

Daube G., Simon P., Limbourg B., Manteca C., Mainil J. and Kaeckenbeeck A. Hybridization of 2659 Clostridium perfringens isolates with gene probes for seven toxins (alpha, beta, epsilon, iota, theta, mu and enterotoxin) and for sialidase. Am J Vet Res, 1996, 57: 496-501.

Katayama S., Dupuy B., Daube G., China B. and Cole S.T. Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne. Mol Gen Genetics, 1996, 251: 720-726.

"Attaching and Effacing"Escherichia coli have been described as a cause of diarrhea in human and in calves. Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli infections are characterised by the formation of "attaching and effacing" lesions on intestinal epithelial cells. The objectives of this project were fundamentals but also applied. The first aim was to further understand the pathogenesis of EPEC and EHEC strains in calves and in dogs. The other aim was to develop diagnostic tools allowing to compare strains isolated from diarrheic and healthy calves and also to compare bovine, human, and canine strains.

The molecular pathogenesis of EPEC and EHECwas mainly studied in human enteropathogenic E. coli strain E2348/69 in which the virulence correlates with the presence of a 35 kb pathogenesis island called LEE. We showed that several strains isolated from calves with diarrhea were able to produce A/E lesions in a rabbit ileal loop model and that they possessed a pathogenesis island related to the LEE. Moreover, we showed that the LEE from bovine strains was mainly inserted at a differnet position in the chromosome compared to the human enteropathogenic E. coli strain E2348/69 (selC gene). In ordre to determine a new site of insertion, we cloned the LEE of a bovine EHEC and sequenced both extremities. In this strain, the LEE doesn't seem to be inserted near a tRNA gene, but the results didn't allow us to determine the exact insertion site.

Virulence genes located in the LEE include the eae, tir, espA and espB genes. These genes have been sequenced from several EPEC and EHEC strains. The sequences alignments revealed the presence of constant and variable regions. Multiplex PCR were developed, in order to determine the subtype of each gene present in a particular isolate. EPEC and EHEC strains isolated from calves dead of diarrhoea, from healthy calves and from infected humans were compared. The same pathotypes were found in sick and healthy calves but in inverted proportion. These pathotypes were also found in human strains. Nevertheless, the human and bovine EHEC strains from serotype O157 possessed their own pathotype.

Secretion extracellular proteins (EspA, EspB, and EspD) via a type III secretion apparatus is necessary for the formation of the A/E lesions by human EPEC and EHEC. As the functional part of this study, we showed that bovine EPEC and EHEC are also able to secrete polypeptides homologous to the already described Esp proteins, most probably via a type III secretion system. Bovine strains present two different secretion profiles of Esp proteins which correlate to the pathotypes of the esp genes as determined by PCR. We also demonstrated that the genes encoding the secreted proteins, present in the LEE of two bovine strains, are organised in the same way as in the human EPEC strain E2348/69.

Enteropathogenic Escherichia coli (EPEC) are isolated from man and ruminants but also from dogs and cats. Dog and cat EPEC isolates were investigated for the presence of plasmidic and chromosomic elements involved in the pathogenesis of EPEC infection, for the production of the A/E lesion in vivo, and for the determination of the biotypes and serotypes. A gene related to the initial adhesion factor (BFP) for human EPEC was detected in a minority of the isolates, in association with high molecular weight plasmids, and a LEE was present in all isolates. As for bovine strains, the LEE was mainly not inserted in the selC region. The eae, tir, espA and EspB genes were analyzed by multiplex PCR. All these genes were present in the tested isolates with heterogeneity in the gene subtypes present. The canine and feline EPEC form a heterogeneous group and some of them are closely related to human EPEC.

In conclusion, this study showed that: (i) the LEE is present in bovine, canine and feline EPEC and EHEC strains; (ii) the LEE is not always inserted at the same site than in human strains; (iii) most of the virulence genes are present in the LEE of bovine and canine strains; (iv) the LEE of bovine and canine strains is functional in vitro and in vivo.

Goffaux F., Mainil J., Pirson V., Charlier G., Pohl P., Jacquemin E., China B. Bovine attaching and effacing Escherichia coli possess a pathogenesis island related to the LEE of the human enteropathogenic Escherichia coli strain E2348/69. FEMS Microbiol Lett, 1997, 154: 415-421.

China B., Goffaux F., Pirson V., Mainil J. Comparison of eae , tir , espA and espB genes of bovine and human attaching and effacing Escherichia coli by muliplex polymerase chain reactions. FEMS Microbiol Lett, 1999, 178: 177-182.

Mainil J., Janssen L., Charlier G., Jacquemin E., China B., Goffaux F. Les souches pathogènes d'Escherichia coli chez les chiens et les chats: II. Données cliniques et bactériologiques sur les souches entéropathogènes. Ann Méd Vét, 2000, 144: 335-343.

Goffaux F., China B., Janssen L., Mainil J. Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats. Res Microbiol, 2000, 151: 865-871.

Goffaux F., China B., Mainil J. Organisation and in vitro expression of esp genes of the LEE (locus of enterocyte effacement) of bovine enteropathogenic and enterohemorrhagic Escherichia coli . Vet Microbiol, 2001, 83: 275-286.

Mycoplasmas frequently infect cattle, causing especially respiratory diseases. Mycoplasma bovis is the most important pathogenic species in countries free of bovine contagious pleuropneumonia. This species was frequently isolated in Belgium from cattle with respiratory disease ante mortem (35 to 50%) and post mortem (20%) in contrast to healthy cattle (0%) and macroscopically healthy lungs (2%). Furthermore, associations were often observed with other mycoplasmas, pasteurellas, Arcanobacterium pyogenes and the bovine respiratory syncytial virus. These isolations were performed from fluids of bronchoalveolar lavage, which is a useful technique to isolate M. bovis from the lower respiratory tract of cattle in field conditions. Of these isolates, many were resistant to several antimicrobial agents which are used in cattle practice. Indeed, high minimum inhibitory concentrations were observed to tetracyclines, macrolides, aminoglycosides and lincosamides suggesting the acquisition of antibiotic resistance in recent Belgian M. bovis isolates.

Inasmuch the high frequency of M. bovis isolation and of antibiotic resistances, it is very important to understand the pathogenicity of this bacteria in order to optimize prophylactic tools. Therefore, the study of the cytadherence of M. bovis is essential since it represents the first step of the bacterial infection. Previous published studies focused on the type strain PG45 which was isolated, in 1962, from milk of a cow with mastitis and subcultured many times in broth medium. However, according to our experimental results, PG45 is not representative of field isolates because of its low adherence rates to various cell lines. This could be explained by the high number of subcultures of the type strain in contrast to other isolates, but not by the origin of the strain (mastitis). Indeed, no significant difference was observed between adherence rates of recent field isolates from arthritis, mastitis and bronchopneumonia. A recent Belgian M. bovis isolate from a pneumonic lung and subcultured only 7 times was further studied to identify the structures of M. bovis conferring adhesion onto bovine bronchial eptihelial cells in primary culture. M. bovis adhered specifically to these cells. Proteins are involved in this step as observed by decreased adherence rates after trypsinization of mycoplasma cells. Additionally, two monoclonal antibodies specific of variable surface lipoproteins, Vsps C and F, significantly reduced the adherence to the primary culture. Other proteins could also be responsible for the adherence. Indeed, we identified by two dimensional electrophoresis a new Vsp that is also involved in the adherence to bovine bronchial epithelial cells. These data comfort the complexity of the M. bovis cytadherence and the involvement of multiple proteins.

Linden A., Thomas A., Mainil J. Les mycoplasmes respiratoires des bovins: I. Clinique, diagnostic et traitement. Ann Méd Vét, 1998, 142: 397-404.

Mainil J., Thomas A., Linden A. Les mycoplasmes respiratoires des bovins: II. Propriétés de virulence et vaccination. Ann Méd Vét, 1998, 142: 405-410.

Thomas A., Dizier I., Trolin A., Mainil J. and Linden A. Comparison of sampling procedures for isolating pulmonary mycoplasmas in cattle. Vet Res Communication, 2002, 26: 333-339.

Thomas A., Ball H., Dizier I., Trolin A., Bell C., Mainil J. and Linden A. Isolation of Mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium. Vet Rec, 2002, 151: 472-476.

Thomas A., Nicolas C., Dizier I., Mainil J. and Linden A. Antibiotic susceptibilities of recent Belgian Mycoplasma bovis isolates. Vet Rec, 2003, 153: 428-431.

Thomas A., Sachse K., Dizier I., Grajetzki C., Farnir F., Mainil J.G. and Linden A. Adherence to various host cell lines of Mycoplasma bovis strains differing in pathogenic and cultural features. Vet Microbiol, 2003, 91: 101-113.

Thomas A., Sachse K., Farnir F., Dizier I., Mainil J. and Linden A. Adherence of Mycoplasma bovis to bovine bronchial eptihelial cells. Microb Pathog, 2003, 34: 141-148.

Thomas A., Mainil J. and Linden A. Mycoplasma bovis: synthèse des connaissances actuelles. Ann Méd Vét, 2003, 147: 23-39.

Thomas A., Dizier I., Sachse K., Ball H., Mainil J. and Linden A. Mycoplasma bovis dans le complexe respiratoire bovin et propriétés de cyto-adhésion in vitro. Ann Méd Vét, 2003, 147: 267-272.

Thomas A., Leprince P., Dizier I., Ball H.J., Gevaert K., Van Damme J., Mainil J. and Linden A. Identification by two-dimensional electrophoresis of a new adhesin expressed by a low-passaged strain of Mycoplasma bovis. Res Microbiol, 2005, 156: 713-718.

During the 1960s, a sudden death syndrome was observed in the suckling beef calf production system in Wallonia (Southern Belgium). Because of the suddenness of the clinical signs and of the lesions of haemorrhagic enteritis at necropsy, the syndrome has been named "red colics" or "torsion" by farmers and "enterotoxaemia" by veterinarians.

The purpose of this work on the "bovine enterotoxaemia syndrome" in suckling calves was: (i) to describe more accurately the clinical signs, macroscopic and microscopic lesions; (ii) to understand the circumstances of occurrence and its actual aetiology; and (iii) to propose a vaccination protocol.

The first study described 78 cases of "bovine enterotoxaemia" in Belgian Blue cattle in Wallonia. Most of the calves were suckling calves from 2 to 4 months of age in excellent health and fattening condition. The clinical signs were a sudden death with much more rarely colics and/or nervous disorders. However even in the latter case the death followed very rapidly. The case history mentioned the existence of various stressful conditions 24 to 36 hours prior to the death. The main, and more consistent, lesion was a generalised, but sometimes, localised necrotic and haemorrhagic enteritis of the jejunum that can extend to the colon. The intestinal content was liquid and haemorrhagic. Acute lymphadenitis was also very common. Histologically the presence of necrotic enteritis was confirmed. These findinds and observations were consistent with an enterotoxaemia syndrome.

During a second study the aerobic and anaerobic intestinal flora of the same population of 78 cases of enterotoxaemia and of a second popoulation of 64 calves that died in other circumstances were compared qualitatively and quantitatively. Clostridium perfringens was isolated in higher numbers (mean values 10 ^7/7,5 versus 10 ^4/5 colony forming units per ml of intestinal content) and from more animals (79 versus 19%) in the "enterotoxaemia" population than in the control population. No other aerobic or anaerobic bacteria could be statistically associated with the "enterotoxaemia" syndrome. All C. perfringens isolates belonged to the non-enterotoxigenic A toxintype, but the gene coding the beta2 toxin was detected by colony hybridisation in 30% of them in either calf population.

During a third study lesions of necrotic and haemorrhagic enteritis were reproduced with various intensity in a ligated intestinal loop assay in one calf with field isolates of C. perfringens belonging to different toxintypes. The results of this assay and of the typing of the C. perfringens isolates rose the hypothesis of a synergistic role between the alpha and beta2 toxins, in 60% of cases of "enterotoxaemia" in suckling calves in Belgium.

In the last study, antibody titres to the alpha toxin were followed after vaccination of the calf and/or of the dam with two commercial vaccines used in Belgium, to optimise a vaccination protocol. The highest titres were obtained in 2 to 4 month-old calves using a first injection at one month of age followed by a booster four weeks later. Further boosters were necessary twice a year to maintain the levels of antibody. High colostral antibody titres can also be obtained by vaccination of the dam at seven and eight months of pregnancy. The colostral antibody can protect the very young calf, but may not be compatible with the above described vaccination protocol designed to obtain a high antibody titre between 2 to 4 months of age. Antibody titres to the beta2 toxin could not be followed at the time of the experiment, because of the very recent description of this new toxin.

Manteca C. and Daube G. L'entérotoxémie en Belgique: I) Introduction et contexte bibliographique. Ann Méd Vét, 1994, 138: 155-164.

Manteca C. and Daube G. L'entérotoxémie en Belgique: I) Introduction et contexte bibliographique. Ann Méd Vét, 1994, 138: 155-164.

Manteca C. and Kaeckenbeeck A. Des postulats de Koch à l'entérotoxémie bovine: petites histoires et vieux papiers. Ann Méd Vét, 2000, 144, 155-164.

Manteca C., Daube G., Jauniaux T., Limbourg B., Kaeckenbeeck A. and Mainil J. Etude de l'entérotoxémie bovine en Belgique. II) Epizootiologie élémentaire et pathologie descriptive. Ann Méd Vét, 2000, 144: 75-82.

Manteca C., Daube G., Pirson V., Limbourg B., Kaeckenbeeck A. and Mainil J.G. Bacterial intestinal flora associated with enterotoxaemia. Vet Microbiol, 2001, 81: 21-32.

Manteca C., Daube G., Jauniaux T., Linden A., Pirson V., Detilleux J., Ginter A., Coppe P., Kaeckenbeeck A. and Mainil J.G. A role for the Clostridium perfringens beta2 toxin in bovine enterotoxaemia? Vet Microbiol, 2002, 86: 191-202.

Manteca C., Ginter A., Limbourg B., Coppe P., Mainil J. and Daube G. Etude de l'entérotoxémie bovine: III) Comparaison de différents protocoles d'immunisation contre la toxine alpha de Clostridium perfringens. Ann Méd Vét, 2004, 148: 147-152.

Necrotoxigenic Escherichia coli (NTEC) are defined on the basis of production of a toxin, the cytotoxic necrotizing factor (CNF) causing the formation of giant multinucleated cells in HeLa cell cultures and necrosis in the rabbit skin. Two different types of CNF have actually been reported: CNF1 which is chromosomally encoded and CNF2 which is coded by a gene located on a plasmid named Vir. CNF1 and CNF2 producing E. coli were called NTEC1 and NTEC2 respectively.

NTEC2 strains have been isolated from various ruminant diseases (gastrointestinal tract infections, pneumonia, septicaemia, metritis, abortion) but have also been isolated from healthy bovines.

In vitro, the interaction of HeLa cells with NTEC2 strains causes the appearance of a cytopathic effect characterized by an enlargement of the cells, a cell cycle arrest in the G2/M phase of the division and the formation of stress fibres. In this model, the actin cytoskeleton reorganization with the appearance of stress fibres is mediated by the CNF2 toxin whereas the cell cycle arrest is caused by a member of the cytolethal distending toxins, CDT-III. The cdt-III gene cluster, made up of the three genes cdt-A, cdt-B and cdt-C, is also located on the Vir plasmid.

In 1975, a calf was inoculated with a strain later identified as NTEC2. This strain caused the death of the animal after appearance of nervous symtpoms and was isolated from blood and internal organs. Since then, no experiment was carried out in order to study the role of NTEC2 strains and CNF2 and CDT-III toxins in disease in calves.

In the first study, 11 colostrum-restricted calves were orally inoculated at 6 hours of age with 2 NTEC2 strains. The first strain (1404) was isolated from the blood of a septicaemic calf. The second strain (B20a) was isolated from the faeces of a diarrheic calf. All these calves presented a liquid and abondant diarrhoea which lasted until euthanasia in 8 of them. In these calves, diarrhoea was correlated with the faecal excretion of the inoculated strains. At necropsy, lesions were: vascular congestion of the intestinal mucosa and hypertrophy of the associated mesenteric lymph nodes. Bacteriology confirmed the colonization of the intestine and the presence of the challenge strains in the heart blood and organs of these calves. The histological lesions were: enterocolitis with lymphadenitis and limited bronchopneumonia.

In the same way, 4 calves were inoculated with a strain identified originally as non-pathogenic (H209) into which the Vir plasmid of the strain 1404 was transferred. This resulted in diarrhoea, intestinal lesions and invasion identical to those observed in wild-type strain inoculated calves.

In order to analyze the respective roles of the toxins CNF2 and the CDT-III, calves were inoculated with isogenic mutants DeltaCNF2 and DeltaCDT-III of the strain 1404. Three of the 4 calves inoculated with the mutant strain 1404DeltaCNF2 did not present any clinical sign in spite of successful intestinal colonization. Nevertheless, the mutant strain remains able to invade the organism and to reach the internal organs of these calves. The calves inoculated with the mutant strain 1404DeltaCDT-III evolved in the same way as wild-type strain inoculated calves with regard to clinical sings, intestinal colonization, microscopic lesions and macroscopic lesions.

Our results suggest that NTEC2 strains are able to colonise the intestine, to cause persistent diarrhea, to invade the blood stream and to localise in the internal organs in colostrum-restricted newborn calves. The CNF2 toxin may play a role in the occurrence of the diarrhea, in contrast to CDT-III, but neither in the development of bacteremia nor in the survival in the blood stream and internal organs.

This work also permitted to confirm the existence of strains NTEC1 and NTEC2 among E. coli strains isolated from diarrheic and septicaemic calves in the years 1960 and 1970 and to confirm the existence, at that time, of the virulence factors associated with these strains: CNF1, CNF2 and CDT-III toxins, as well as F17, Pap, Afa-VIII and Sfa adhesins.

Lastly, we developed two multiplex PCR allowing the detection of genes coding for the above mentioned virulence factors and the aerobactin.

Van Bost S. and Mainil J. Reproduction of lesions and clinical sings with a CNF2-producing Escherichia coli in neonatal calves. Adv Exp Med Biol, 1999, 473: 125-128.

Van Bost S., Babê M.H., Jacquemin E., Mainil J. Characteristics of necrotoxigenic Escherichia coli isolated from septicemic and diarrheic calves between 1958 and 1970. Vet Microbiol, 2001, 82: 311-320.

Van Bost S., Roels S., Mainil J. Necrotoxigenic Escherichia coli type-2 invade and cause diarrhoea during experimental infection in colostrum-restricted newborn calves. Vet Microbiol, 2001, 81: 315-329.

Van Bost S., Roels S., Oswald E., Mainil J. Putative roles of the CNF2 and the CDT-III toxins in experimental infections with necrotoxigenic Escherichia coli type 2 (NTEC2) strains in calves. Microbiol Infect, 2003, 5: 1189-1193.

Van Bost S., Jacquemin E., Oswald E., Mainil J. Multiplex PCRs for identification of necrotoxigenic Escherichia coli. J Clin Microbiol, 2003, 41: 4480-4482.

Van Bost S. and Mainil J. Facteurs de virulence spécifiques des souches invasives d'Escherichia coli: III) Production de toxines. Ann Méd Vét, 2003, 147: 327-342.

The species E. coli is highly heterogeneous, including pathogenic and non-pathogenic strains. The classification of the pathogenic strains is made upon specific virulence factors. Some of them are not capable to cross over the intestinal border and are responsible for intestinal disorders associated with diarrhoea. Other strains can cross the intestinal border and produce septicaemia and other complications depending on the infected organs. These pathogenic strains of E. coli interact with the host in a typical manner and produce specific lesions. These specific interactions are observed after the colonisation of the gut by pathogenic bacteria and are the results of the presence of specific virulence factors. The colonisation of the gut is mediated by specific adhesins, which are specific for each group of pathogenic E. coli. It seems that these adhesins are not only responsible to initiate the interaction between the pathogen and the host but are also responsible for the host specificity shown by some pathogenic strains. It is the reason that a better understanding of these adhesins would permit a better understanding of the host specificity and a better evaluation fo the zoonotic potential of these strains.

The aim of this work was to identify bacterial structures involved in the intestinal adhesion step and in the intestinal colonisation of the enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) and verotoxigenic E. coli (VTEC) bovine isolates, focusing mainly (but not exclusively) on strains of serogroup O26.

In order to achieve this objective, two different approaches have been followed: i) the immunologic approach and ii) the genetic approach. The immunologic approach was using the benefits of the 2F3 monoclonal antibody (MAb), which was obtained against an outer membrane protein extract from a human O26 EHEC strain. The work on this approach focused on the study of this MAb as epidemiological tool and on the identification of the epitope recognised by this antibody and of its genetic basis.

The work on the genetic approach involved the screening by PCR and by colony hybridization of a collection of EHEC, VTEC and EPEC of human and bovine isolates for the presence of homologues for gene clusters coding for fimbriae of type I, II, III and IV: i) putative adhesins of human EPEC and EHEC strains like LifA, Iha, LpfA, BfpA; ii) fimbrial adhesins (CS31A et F17) and afimbrial adhesins of Afa family (Afa III, Afa VII, Afa VIII and F1845) described for other groups of bovine pathogenic E. coli.

The accomplished work allowed, first of all, to confirm that the 2F3 MAb is specific of the O26 EPEC and O26 EHEC strains without being capable to distinguish between human and animal isolates, that means without being capable to distinguish them depending on their host.

Moreover, it has been shown that: i) the epitope recognised by the 2F3 MAb is a component of the O antigen of EPEC and EHEC strains; ii) the genetic basis of this epitope is located inside the O antigen gene cluster of O26 EPEC and O26 EHEC strains; and iii) the 2F3 + and O26 + characters are two characters that can be dissociated by random insertional mutagenesis in a bovine EHEC strain. Nevertheless, the results of this work did not allow us to explain the specificity of the 2F3 MAb for the O26 EPEC and O26 EHEC strains. Actually, by sequencing the O-antigen gene cluster (the rfb locus) of an O26 non-EPEC and non-EHEC strain and comparing the obtained sequence with the already published sequence of the O-antigen gene cluster of an O26 EHEC strain no major difference (which could explain the specificity of the 2F3 MAb for the O26 EPEC or EHEC strains) was observed.

The obtained results in the genetic approach showed that: i) all O145 and O157 EPEC and EHEC strains were positive for the LpfA probe (lpfA gene is coding for a type IV pili) and all other human and bovine strains belonging to other serogroups (O5, O26, O103, O111 and O118) tested negative; ii) a large majority of EPEC and EHEC strains belonging to O5, O26, O103 or O118 serogroups were positive for the LifA probe (the lifA gene is involved in the synthesis of still not very well characterized adhesins), but none of those belonging to the O145 and O157 serogroups; iii) different proportions of these strains belonging to various serogroups were positive for the Iha probe (the Iha gene is involved in the synthesis of a not very well characterized adhesin); and iv) ten out of twelve bovine O26 EPEC strains were positive for the ClpE probe and all other strains (human EPEC and EHEC strains and bovine EHEC strains) were negative for the ClpE probe. The clpE gene is involved in the synthesis of the chaperon protein of the CS31A fimbriae, which was described on enterotoxigenic and septicaemic bovine and porcine E. coli isolates.

Since the O145 and O157 EHEC strains show the same pathotype, it looks like that the presence of the lpfA gene and the absence of the lifA gene is more linked to a specific pathotype than to a specific serotype. Concerning the results obtained with the ClpE probe for the O26 EPEC strains, it is possible that these strains have not a truncated clp gene cluster since all the strains positive for the ClpE probe were negative for the ClpG (clpG gene is involved in the biosynthesis of the major unit of the CS31A fimbriae). Nevertheless, it is possible that the clpE-like gene of these strains belongs to an entire gene cluster for which the major unit would be different from the ClpG.

Finally, the screening of a collection of O8 and O20 VTEC bovine isolates for the presence of sequences homologous to the genes involved in the biosynthesis of adhesions of the Afa family (Afa III, Afa VII, Afa VIII and F1845), produced mainly by human uropathogenic isolates and by some septicaemic strains isolated from animals, allowed the identification of two O8 VTEC bovine strains harbouring the afa-8D/afa-8E genes and expressing the Afa VIII E adhesin.

Szalo I.M., Goffaux F., Pirson V., Piérard D., Ball H.J., Mainil J. Presence in bovine enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli of genes encoding for putative adhesins of human EHEC strains. Res. Microbiol., 2002, 153: 653-658.

Szalo I.M., Taminiau B., Goffaux F., Pirson V., McCappin J., Ball H.J., Mainil J. The 2F3 monoclonal antibody recognises the O26 O-antigen moiety of the lipopolysaccharide of enterohaemorrhagic Escherichia coli strain 4276. Clin Diagn Lab Immunol, 2004, 11: 532-537.

Szalo M., Taminiau B., Mainil J. Le lipopolysaccharide d'Escherichia coli: structure, biosynthèse et rôles. Ann Méd Vét, 2006, 150: 108-124.