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Escherichia coli was described in 1885 by a German pediatrician, Theodor Escherich, in the faeces of a child suffering diarrhoea. In 1893, a Danish veterinarian postulated that the E. coli species comprises different strains, some being pathogens, others not. Today the E. coli species is subdivided into several pathogenic strains causing different intestinal, urinary tract or internal infections and pathologies, in animal species and in humans. Since this congress topic is the interaction between E. coli and the mucosal immune system, the purpose of this manuscript is to present different classes of adhesins (fimbrial adhesins, afimbrial adhesins and outer membrane proteins), the type 3 secretion system, and some toxins (oligopeptide, AB, and RTX pore-forming toxins) produced by E. coli, that can directly interact with the epithelial cells of the intestinal, respiratory and urinary tracts., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: One method to improve durably animal welfare is to select, as reproducers, animals with the highest ability to resist or tolerate infection. To do so, it is necessary to distinguish direct and indirect mechanisms of resistance and tolerance because selection on these traits is believed to have different epidemiological and evolutionary consequences., Methods: We propose structural equation models with latent variables (1) to quantify the latent risk of infection and to identify, among the many potential mediators of infection, the few ones that influence it significantly and (2) to estimate direct and indirect levels of tolerance of animals infected naturally with pathogens. We applied the method to two surveys of bovine mastitis in the Walloon region of Belgium, in which we recorded herd management practices, mastitis frequency, and results of bacteriological analyses of milk samples., Results and Discussion: Structural equation models suggested that, among more than 35 surveyed herd characteristics, only nine (age, addition of urea in the rations, treatment of subclinical mastitis, presence of dirty liner, cows with hyperkeratotic teats, machine stripping, pre- and post-milking teat disinfection, and housing of milking cows in cubicles) were directly and significantly related to a latent measure of bovine mastitis, and that treatment of subclinical mastitis was involved in the pathway between post-milking teat disinfection and latent mastitis. These models also allowed the separation of direct and indirect effects of bacterial infection on milk productivity. Results suggested that infected cows were tolerant but not resistant to mastitis pathogens., Conclusions: We revealed the advantages of structural equation models, compared to classical models, for dissecting measurements of resistance and tolerance to infectious diseases, here bovine mastitis. Using our method, we identified nine major risk factors that were directly associated with an increased risk of mastitis and suggested that cows were tolerant but not resistant to mastitis. Selection should aim at improved resistance to infection by mastitis pathogens, although further investigations are needed due to the limitations of the data used in this study.

In order to improve the identification of avian pathogenic Escherichia coli (APEC) strains, an extensive characterization of 1,491 E. coli isolates was conducted, based on serotyping, virulence genotyping, and experimental pathogenicity for chickens. The isolates originated from lesions of avian colibacillosis (n = 1,307) or from the intestines of healthy animals (n = 184) from France, Spain, and Belgium. A subset (460 isolates) of this collection was defined according to their virulence for chicks. Six serogroups (O1, O2, O5, O8, O18, and O78) accounted for 56.5% of the APEC isolates and 22.5% of the nonpathogenic isolates. Thirteen virulence genes were more frequently present in APEC isolates than in nonpathogenic isolates but, individually, none of them could allow the identification of an isolate as an APEC strain. In order to take into account the diversity of APEC strains, a statistical analysis based on a tree-modeling method was therefore conducted on the sample of 460 pathogenic and nonpathogenic isolates. This resulted in the identification of four different associations of virulence genes that enables the identification of 70.2% of the pathogenic strains. Pathogenic strains were identified with an error margin of 4.3%. The reliability of the link between these four virulence patterns and pathogenicity for chickens was validated on a sample of 395 E. coli isolates from the collection. The genotyping method described here allowed the identification of more APEC isolates with greater reliability than the classical serotyping methods currently used in veterinary laboratories.

Staphylococcus (S.) aureus is a major pathogen involved in chronic bovine mastitis. Staphylococcal mastitis is difficult to control due to the ability of S. aureus to invade and survive within host cells. We therefore postulated that induction of CD8(+) cytotoxic T lymphocyte (CTL) responses leading to destruction of infected cells could help in the control of S. aureus mastitis. We demonstrate that immunization of mice with heat-killed S. aureus together with agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of reducing the severity of subsequent staphylococcal mastitis. Our study shows promise for CTL-dependent vaccination against S. aureus mastitis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

An enteroaggregative Verotoxin (Vtx)-producing Escherichia coli strain of serotype O104:H4 has recently been associated with an outbreak of haemolytic-uremic syndrome and bloody diarrhoea in humans mainly in Germany, but also in 14 other European countries, USA and Canada. This O104:H4 E. coli strain has often been described as an enterohaemorrhagic E. coli (EHEC), i.e. a Vtx-producing E. coli with attaching and effacing properties. Although both EHEC and the German O104:H4 E. coli strains indeed produce Vtx, they nevertheless differ in several other virulence traits, as well as in epidemiological characteristics. For instance, the primary sources and vehicles of typical EHEC infections in humans are ruminants, whereas no animal reservoir has been identified for enteroaggregative E. coli (EAggEC). The present article is introduced by a brief overview of the main characteristics of Vtx-producing E. coli and EAggEC. Thereafter, the O104:H4 E. coli outbreak is compared to typical EHEC outbreaks and the virulence factors and host specificity of EHEC and EAggEC are discussed. Finally, a renewed nomenclature of Vtx-producing E. coli is proposed to avoid more confusion in communication during future outbreaks and to replace the acronym EHEC that only refers to a clinical condition.

Recent studies have shown that hydro-alcoholic solutions are more efficient than traditional medicated soaps in the pre-surgical hand antisepsis of human surgeons but there is little veterinary literature on the subject. The aim of this study was to compare the efficiency of medicated soaps and a hydro-alcoholic solution prior to surgery using an in-use testing method in a veterinary setting. A preliminary trial was performed that compared the mean log(10) number of bacterial colony forming units (CFU) and the reduction factors (RF) between two 5-min hand-scrubbing sessions using different soaps, namely, povidone iodine (PVP) and chlorhexidine gluconate (CHX), and the 1.5-min application of a hydro-alcoholic rub. A clinical in-use trial was then used to compare the hydro-alcoholic rub and CHX in a surgical setting. Sampling was performed using finger printing on agar plates. The hydro-alcoholic rub and CHX had a similar immediate effect, although the sustained effect was significantly better for the hydro-alcoholic rub, while PVP had a significantly lower immediate and sustained effect. The hydro-alcoholic rub showed good efficiency in the clinical trial and could be considered as a useful alternative method for veterinary surgical hand antisepsis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

As neglected zoonosis for many years, Q fever is now ubiquitous in Japan. Similarly to elsewhere in the world, domestic animals are considered to be important reservoirs of the causal agent, Coxiella burnetii, a resistant intracellular bacterium. Infected animals shed bacteria in milk, feces, urine, vaginal mucous and birth products. Inhalation of bacteria present in the environment is the main route of animal and human infection. Shedding of C. burnetii in milk by domestic ruminants has a very limited impact as raw milk is seldom ingested by the Japanese population. The clinical expression of Q fever in Japan is similar to its clinical expression elsewhere. However clinical cases in children are more frequently reported in this country. Moreover, C. burnetii is specified as one of the causative organisms of atypical pneumonia in the Japanese Respiratory Society Guideline for the management of community-acquired pneumonia. In Japan, C. burnetii isolates are associated with acute illness and are mainly of moderate to low virulence. Cats are considered a significant source of C. burnetii responsible for human outbreaks in association with the presence of infected parturient cats. Since its recognition as a reportable disease in 1999, 7-46 clinical cases of Q fever have been reported by year. The epidemiology of Q fever in Japan remains to be elucidated and the exact modes of transmission are still unproven. Important further research is necessary to improve knowledge of the disease itself, the endogenous hosts and reservoirs, and the epidemiological cycle of coxiellosis in Japan., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Epizootic rabbit enteropathy (ERE) emerged and spread in Europe within the last 13 years causing major economical loss. The aims of the study was to evaluate antibiograms of TEC4, an inoculum composed of an extract of intestinal content of affected rabbits, and to test the potential of different antibiotic-based TEC4 fractions to reproduce the disease. Twenty nine different antibiotic discs were incubated for determining bacteria resistance. In a complementary study, nine tubes of liquid medium were inoculated with TEC4, incubated and added individually with amoxicillin/clavulanic acid, bacitracin, ceftiofur, doxycycline, novobiocin, streptomycyin, tylosin, vancomycin and 0.9% saline solution as control. The content of each tube was washed by centrifugation and suspended in saline. The three most effective antibiotics are florfenicol, amoxycillin/clavulanic acid and tylosin. A high concentration of Clostridium sordelli and Bacillus firmus were isolated in all fractions. Species never cultured from TEC4 were identified as Fusobacterium necrogenes (in vancomycin fraction), Cellulomonas sp (in novobiocin fraction) and Bacteroides distasonis (in doxycycline fraction). The ERE was reproduced when bacitracin, doxycycline and 0.9% fractions were inoculated. Rabbits showed ERE clinical signs with the specific drop in daily weight gain.

Q fever is an ubiquitous zoonosis caused by an resistant intracellular bacterium, Coxiella burnetii. In certain areas, Q fever can be a severe public health problem, and awareness of the disease must be promoted worldwide. Nevertheless, knowledge of Coxiella burnetii remains limited to this day. Its resistant (intracellular and environmental) and infectious properties have been poorly investigated. Further understanding of the interactions between the infected host and the bacteria is necessary. Domestic ruminants are considered as the main reservoir of bacteria. Infected animals shed highly infectious organisms in milk, feces, urine, vaginal mucus, and, very importantly, birth products. Inhalation is the main route of infection. Frequently asymptomatic in humans and animals, Q fever can cause acute or chronic infections. Financial consequences of infection can be dramatic at herd level. Vaccination with inactive whole-cell bacteria has been performed and proved effective in humans and animals. However, inactive whole-cell vaccines present several defects. Recombinant vaccines have been developed in experimental conditions and have great potential for the future. Q fever is a challenging disease for scientists as significant further investigations are necessary. Great research opportunities are available to reach a better understanding and thus a better prevention and control of the infection.

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) inject a repertoire of effector proteins into host cells via a type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE). OspG is an effector protein initially identified in Shigella that was shown to inhibit the host innate immune response. In this study, we found ospG homologues in EHEC (mainly of serogroup O111) and in Yersinia enterocolitica. The T3SS encoded by the LEE was able to inject these different OspG homologues into host cells. Infection of HeLa cells with EHEC O111 inhibited the NF-kappaB-dependent innate immune response via a T3SS-dependent mechanism. However, an EHEC O111 ospG mutant was still able to inhibit NF-kappaB p65 transfer to the nucleus in infected cells stimulated by tumour necrosis factor alpha (TNF-alpha). In addition, no difference in the inflammatory response was observed between wild-type EHEC O111 and the isogenic ospG mutant in the rabbit ligated intestinal loop model. These results suggest that OspG is not the sole effector protein involved in the inactivation of the host innate immune system during EHEC O111 infection.

In the present study we developed an enzymatic approach (through the use of collagenase and dispase) to isolate bovine intestinal epithelial cells. Using this method, freshly isolated jejunocytes could be distinguished from simultaneously isolated colonocytes, as the jejunocytes specifically exhibited the small intestinal peptidase gene transcript, as well as an active alkaline phosphatase. The transformation of both types of cell suspension was performed by retroviral infection, using reproduction-defective viruses bearing the gene coding for the large T antigen of the leukaemia simian virus (SV40). The success of the transfection was demonstrated by (1) a significant increase in cell passage numbers (52-53 vs. 7 passages for non-transfected cells), (2) the detection of both the large T transcript and the large T antigen in transformed cells. Possible contamination and progressive substitution of bovine primocultures by non-bovine lineages available in the laboratory was excluded, as the transformed cells presented a bovine typical karyotype. Most transfected cells kept an epithelial morphology after transformation. They also maintained the expression of FABP and enterocyte specific enzymes (brush-border associated maltase and IAP). However, levels of specific activity of these enzymes were low, suggesting that cell differentiation is not completely achieved under the applied culture conditions.

Insertion of heterologous peptide sequences into a protein carrier may impose structural constraints that could help the peptide to adopt a proper fold. This concept could be the starting point for the development of a new generation of safe subunit vaccines based on the expression of poorly immunogenic epitopes. In the present study, we characterized the tolerance of the TEM-1 class A beta-lactamase to the insertion of two different peptides, the V3 loop of the gp120 protein of HIV, and the thermostable STa enterotoxin produced by enterotoxic Escherichia coli. Insertion of the V3 loop of the HIV gp120 protein into the TEM-1 scaffold yielded insoluble and poorly produced proteins. By contrast, four hybrid beta-lactamases carrying the STa peptide were efficiently produced and purified. Immunization of BALB/c mice with these hybrid proteins produced high levels of TEM-1-specific antibodies, together with significant levels of neutralizing antibodies against STa.

Overgrowth of Clostridium perfringens clones with production of one or more of its toxin(s) results in diverse digestive and systemic pathologies in human and animals, such as cattle enterotoxaemia. The so-called beta2 toxin (CPB2) is the most recently described major toxin produced by C. perfringens. In this study, the cpb2 ORF (cpb2FM) from a cattle C. perfringens-associated enterotoxaemia was cloned and sequenced. The cpb2FM and its deduced nucleotide sequence clearly corresponded to the cpb2 allele considered as "consensus" and not to "atypical" allele, despite its "non-porcine" origin. Expression assays of the recombinant toxin CPB2FM were performed in Escherichia coli and Bacillus subtilis with the expression vector pBLTS72, and by genomic integration by double recombination in B. subtilis. Highest level of production was obtained with the expression vector in B. subtilis 168 strain. The recombinant CPB2FM protein was purified and a specific rabbit polyclonal antiserum was produced. Polyclonal antibodies could detect CPB2 production in supernatants of C. perfringens from enterotoxaemic cattle.

Clostridium perfringens has been implicated in a broad array of enteric infections including the fatal haemorrhagic enteritis/enterotoxaemia syndrome in cattle. The beta2 toxin (CPB2), encoded by cpb2, is suspected to be implicated in this syndrome. However, among C. perfringens isolates from cattle suspected of clostridial disease, an atypical allele was recently found to predominate at the cpb2 locus and atypical corresponding CPB2 proteins were shown to be poorly expressed, thus arguing against a biologically significant role of the beta2 toxin in clostridial diseases in cattle. This study compared genotype and phenotype of the beta2 toxin between C. perfringens isolates from a group of healthy calves (n=14, 87 isolates) and from a group of enterotoxaemic calves (n=8, 41 isolates). PCR results revealed the exclusive presence of the typical "consensus"cpb2 in the enterotoxaemic group. Western blot analysis demonstrated that the typical variant of CPB2 was often expressed in isolates from enterotoxaemic calves (43.9%) and infrequently in isolates from healthy cattle (6.9%). These data suggest that the typical variant of the CPB2 toxin may play a role in the pathogenesis of cattle enterotoxaemia.

Extraintestinal pathogenic (ExPEC) Escherichia coli strains of serotype O18:K1:H7 are mainly responsible for neonatal meningitis and sepsis in humans and belong to a limited number of closely related clones. The same serotype is also frequently isolated from the extraintestinal lesions of colibacillosis in poultry, but it is not well known to what extent human and avian strains of this particular serotype are related. Twenty-two ExPEC isolates of human origin and 33 isolates of avian origin were compared on the basis of their virulence determinants, lethality for chicks, pulsed-field gel electrophoresis (PFGE) patterns, and classification in the main phylogenetic groups. Both avian and human isolates were lethal for chicks and harbored similar virulence genotypes. A major virulence pattern, identified in 75% of the isolates, was characterized by the presence of F1 variant fimbriae; S fimbriae; IbeA; the aerobactin system; and genomic fragments A9, A12, D1, D7, D10, and D11 and by the absence of P fimbriae, F1C fimbriae, Afa adhesin, and CNF1. All but one of the avian and human isolates also belonged to major phylogenetic group B2. However, various subclonal populations could be distinguished by PFGE in relation to animal species and geographical origin. These results demonstrate that very closely related clones can be recovered from extraintestinal infections in humans and chickens and suggest that avian pathogenic E. coli isolates of serotype O18:K1:H7 are potential human pathogens.

Background: Cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract., Results: Cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein) as well as an epithelial cytoskeleton component (cytokeratin 18). However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker). Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1-2 mM) or using a glucose-deprived culture medium., Conclusion: The present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations.

A significant decrease in adherence rates of Mycoplasma bovis to bovine bronchial epithelial (BBE) cells has been observed after passage of the organism in artificial medium. Analysis of the proteins expressed by M. bovis isolate 2610 by two-dimensional (2-D) electrophoresis demonstrated differences between the cells harvested after the 7th and 116th passage. Three silver-stained prominent spots observed in 2-D electrophoretic separation of protein extracts of the lower-passaged cells were considerably less strongly expressed in the sample from higher-passaged cells. These spots had a molecular mass of approximately 24 kDa and an isoelectric point of about 5. The mass spectrometry analysis of these trypsin-sensitive proteins led to their identification as a unique new member of the Vsps family of membrane-associated proteins. Serum from a mouse immunized with these proteins significantly reduced adherence of M. bovis to BBE cells. This result underlines the function of this new Vsp in adherence of M. bovis to host cells.

In order to test the use of DNA vaccination for its capacity to induce antibodies against the non-immunogenic heat-stable enterotoxin STa from Escherichia coli, BALB/c mice were immunized with plasmid DNA encoding hybrid proteins made by the insertion of wild type STa or insertion of the Cys6Ala, Cys17Ala and Cys6Ala-Cys17Ala STa mutants at positions 195 or 216 of the TEM-1 beta-lactamase. No STa specific antibodies could be detected after three plasmid injections, but a subsequent boost with native STa peptide was capable of inducing low levels of neutralizing antibodies, as tested in the suckling mouse assay. Highest STa specific responses were found in mice primed with the double mutated STa inserted in position 195. This plasmid induced highest T-cell responses to the TEM-1 protein, indicating that priming of helper T-cell responses to the carrier protein was essential. Mixed IgG1/IgG2a isotypes also reflected this T helper 1 type priming. Moreover, insertion into loop A of the TEM-1 carrier may be more suitable than insertion into loop B, because of reduced competition between carrier and hapten B cell responses.

This study aimed to investigate whether Escherichia coli endotoxin (LPS) may predispose the lung to an infection with Pasteurella multocida type A (Pma) and to determine the LPS concentration needed to reproduce clinical signs of bronchopneumonia. Twenty-four hours before inoculating Pma or sterile growth medium, piglets were tracheally instilled with 10, 100 or 400 microg/kg LPS. Cough, body temperature, daily weight gain (DWG) bronchoalveolar lavage fluid (BALF) cells and volume of pneumonic lung were measured. Changes in breathing pattern (Penh) were assessed by whole body barometric plethysmography. No significant changes were observed in Pma-treated or in control animals. Each LPS doses induced DWG reduction while the higher generated a severe subacute interstitial pneumonia causing hyperthermia and an increase in Penh. The combination of the lower LPS doses with Pma produced an asymptomatic bronchopneumonia leading to DWG reduction, rise in Penh and an increase in BALF macrophages and neutrophils. With 400 microg/kg LPS, Pma worsened the inflammatory process as illustrated by cough, hyperthermia, major DWG reduction and by a greater Penh response. Lung lesions consisted of severe exudative bronchopneumonia. We concluded that LPS may negatively influence growth, predispose to persisting lung inflammatory process and promote Pma infection depending on the dose previously administered.

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.

An analogue of the adhesin gene p40 of Mycoplasma agalactiae was found in Mycoplasma bovis. Nucleotide sequence analysis of the p40* gene in M. bovis revealed the presence of a large deletion involving a frameshift that causes premature truncation of the translated protein, indicating that p40* exists as a pseudogene in M. bovis.

Recent studies by DNA-DNA hybridisation assays conducted on a large collection of Escherichia coli strains isolated from chickens, ducks and turkeys suffering from colibacillosis, showed that 76% of the strains were negative for the presence of the pap gene cluster. The objective of this paper was to study the virulence associated with the avian E. coli strains negative for the P fimbriae, but carrying the f17 or the afa-8 gene cluster coding for adhesins associated with strains pathogenic for mammals. Three strains carrying the f17 fimbriae and three carrying the afa-8 adhesin-encoding gene cluster were studied in three in vivo experimental models of avian colibacillosis: subcutaneous inoculation of 1-day-old chicks, inoculation of specific-pathogen-free (SPF) chickens via the intra-thoracic air sac, and intra-tracheal inoculation of axenic chickens. The results showed that the six P-negative E. coli isolates carrying the f17 or the afa-8 gene cluster were lethal for 1-day-old chicks. They were also able to reproduce clinical signs and lesions of colibacillosis (aerosacculitis, pericarditis, perihepathitis), with bacteraemia and septicaemia, in SPF chickens inoculated via the thoracic air sacs as well as in axenic chickens inoculated by the intra-tracheal route. Further studies with f17 and afa-8 allelic mutants constructed by disruption must be performed to confirm a role of F17 fimbrial and Afa-VIII afimbrial adhesins in the pathogenesis of avian colibacillosis.

The aims of this study were (1) to correlate cough and body temperature (BT) with the severity of bronchopneumonia in pigs, (2) to determine whether these clinical signs can be used to early diagnose bronchopneumonia and (3) to assess the predictive values of cough and BT regarding lung lesions. Bronchopneumonia was induced by administering E. coli endotoxin (LPS) combined with Pasteurella multocida type A (PmA) in the trachea of 13 piglets. Saline-instilled negative controls (n = 8), PmA inoculated (n = 6) and LPS instilled (n = 5) groups were also constituted. Cough and BT were recorded daily while the bronchopneumonia severity was assessed using bronchoalveolar lavage fluid (BALF) cytology, cytokines and measurement of lung lesion volume. Changes in expiratory breathing pattern were also measured (Penh). The combination of LPS and PmA induced a subacute bronchopneumonia characterised by macrophage, neutrophil, and lymphocyte infiltration, changes in Penh and an increase in the mRNA level of IFN-gamma while IL8, IL-18 and TNF-alpha mRNA levels remained unchanged. The daily body weight gain of infected animals was significantly reduced. Cough and BT changes were proportional to the intensity of the lung inflammatory process, functional respiratory changes and to the extent of macroscopic lesions. When comparing the individual values of cough and BT to thresholds defined for both parameters, an early diagnosis of pneumonia was possible. Considering the pooled data of each group, it was possible to define thresholds allowing an early segregation between the groups of diseased and healthy piglets. The daily values of cough and BT were predictive for the volume of lung lesions recorded at the end of the trial. In conclusion, cough and BT appear as potential indicators for the intensity and the evolution of the respiratory disease. They also seem to be good predictors for the magnitude of lung lesions and weight gain recorded at the study endpoint., (Copyright 2004 INRA, EDP Sciences)

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) organisms are groups of pathogenic strains whose infections are characterized by a typical lesion of enterocyte attachment and effacement. They are involved in enteric diseases both in humans and in animals, and EHEC strains can be responsible for hemolytic uremic syndrome in humans. Previously, it was shown that the 2F3 monoclonal antibody (MAb) is specific for the O26 EHEC and EPEC strains (P. Kerr, H. Ball, B. China, J. Mainil, D. Finlay, D. Pollock, I. Wilson, and D. Mackie, Clin. Diagn. Lab. Immunol. 6:610-614, 1999). As these groups of bacteria play an important role in pathology, the aim of this paper was to characterize the antigen recognized by the 2F3 MAb and its genetic determinant. A genomic locus containing the entire O-antigen gene cluster and half of the colanic acid gene cluster from an O26 EHEC strain was shown to be sufficient for the production of the antigen recognized by the 2F3 MAb in an E. coli DH5alpha strain. By transposon mutagenesis performed on the recombinant plasmid, all 2F3 enzyme-linked immunosorbent assay-negative mutants had their transposons inserted into the O-antigen gene cluster. The O-antigen gene cluster was also cloned from an O26 EHEC strain into the E. coli DH5alpha strain, which then produced a positive result with the 2F3 MAb. Further analysis of the type of lipopolysaccharides (smooth or rough) produced by the clones and mutants and of the O antigen of the 2F3-positive clones confirmed that the epitope recognized by the 2F3 MAb is located on the O antigen in the O26 EHEC and EPEC strains and that its genetic determinant is located inside the O-antigen gene cluster.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. Both use a type III secretion system (TTSS) encoded by the 'locus of enterocyte effacement' (LEE) to subvert and attach to epithelial cells through the injection of a repertoire of effector molecules. Here, we report the identification of a new TTSS translocated effector molecule called Cif, which blocks cell cycle G2/M transition and induces the formation of stress fibres through the recruitment of focal adhesions. Cif is not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface, but is defective in the ability to block mitosis. When expressed in TTSS competent LEE-positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G2/M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins that subvert the host eukaryotic cell cycle.

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.

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.

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms of cytadherence and the molecular factors involved. The purpose of this work was to compare adherence rates of M. bovis field strains to different host cell lines and study the effects of cloning and sub-culturing M. bovis strains on their adherence properties. Eighteen metabolically labeled M. bovis strains isolated from different pathological backgrounds were examined in adherence trials using four different host cell lines, i.e. embryonic bovine lung (EBL), embryonic bovine trachea (EBTr), Madin Darby bovine kidney (MDBK) and rabbit kidney (RK) cells. Although large interstrain variations in adherence rates (3.4-19.1%) were measured they could not be correlated to the pathological background (pneumonia, arthritis or mastitis). Adherence rates to the fibroblast cell line (EBTr) were significantly lower than those to the three epithelial cell lines (EBL, MDBK and RK). The only non-pathogenic strain (221/89) exhibited lower adherence rates than three isolates from clinical mastitis. Interestingly, adherence rates were significantly reduced after in vitro passaging. In contrast, no effect of single cloning of strains on adherence was observed. There was no general correlation between expression of variable surface proteins (Vsps) as monitored by immunoblotting and adherence rates, although alterations in Vsp expression profiles were seen as a consequence of passaging. As there is probably a large number of adhesins, variable and non-variable, on the surface of M. bovis cells the issue is very complex, and the most active components have yet to be identified.

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) infections are characterised by the formation of attaching and effacing lesions on intestinal epithelial cells. The first step of EPEC and EHEC pathogenesis involves the initial adherence of the bacterium to the intestinal epithelium. A collection of bovine EPEC and EHEC strains belonging to different serogroups was tested by colony blot hybridization with gene probes for putative adhesins (BFPA, LPFA, IHA, LIFA) of human EPEC and EHEC, and also for fimbrial and afimbrial adhesins (AFA8, F17, Cs31A) of bovine necrotoxigenic E. coli (NTEC). In the bovine EPEC and EHEC strains tested, sequences homologous to lifA, ihA, and lpfA genes were detected, sometimes in association with particular serogroups. Bovine 026 EPEC also possessed a sequence homologous to a gene of the c/p operon, coding for the CS31A adhesin, associated with bovine NTEC. Overall results showed that different genes encoding for putative adhesins of human EHEC strains are present in bovine EPEC and EHEC strains, but not one of them is present in all strains.

Non-enterotoxigenic type A Clostridium perfringens are associated with bovine enterotoxaemia, but the alpha toxin is not regarded as responsible for the production of typical lesions of necrotic and haemorrhagic enteritis. The purpose of this study was to investigate the putative role of the more recently described beta2 toxin. Seven hundred and fourteen non-enterotoxigenic type A C. perfringens isolated from 133 calves with lesions of enterotoxaemia and high clostridial cell counts (study population) and 386 isolated from a control population of 87 calves were tested by a colony hybridisation assay for the beta2 toxin. Two hundred and eighteen (31%) C. perfringens isolated from 83 calves (62%) of the study population and 113 (29%) C. perfringens isolated from 51 calves (59%) of the control population tested positive with the beta2 probe. Pure and mixed cultures of four C. perfringens (one alpha+beta2+, one alpha+enterotoxin+ and two alpha+) were tested in the ligated loop assay in one calf. Macroscopic haemorrhages of the intestinal wall, necrosis and haemorrhages of the intestinal content, and microscopic lesions of necrosis and polymorphonuclear and mononuclear cell infiltration of the intestinal villi were more pronounced in loops inoculated with the alpha and beta2-toxigenic C. perfringens isolate. These results suggest in vivo synergistic role of the alpha and beta2 toxins in the production of necrotic and haemorrhagic lesions of the small intestine in cases of bovine enterotoxaemia. However, isolation of beta2-toxigenic C. perfringens does not confirm the clinical diagnosis of bovine enterotoxaemia and a clostridial cell counts must still be performed.

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.

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.

In order to characterize potential pathogenic Escherichia coli strains isolated from diarrheic hens and chickens originating from intensive battery rearing in North Algeria, the presence of a large range of virulence factors and markers was studied in 50 strains by DNA-DNA hybridization on colonies and phenotypic tests. The sequences we focused on were those coding for adhesins F5, F41, F17, Pap, Afa, and Sfa; intimin Eae; and toxins STa, STb, LT1, Stx1, Stx2, CNF1, and CNF2. The phenotypes explored were the colicins, aerobactin, hemolysins, and hemagglutinin production and serum resistance. The genotypic and phenotypic tests enabled us to categorize the isolates into two distinct groups: those with a potential to invade the host (27 strains were serum resistant and/or produced aerobactin), among which three strains were also potentially diarrheagenic, one strain was LT1 + F17+ Afa+ Pap+ (enterotoxigenic E. coli) and the two others were Stx1 (verotoxigenic E. coli). Twenty-three strains were colicinogenic, including 19 strains producing colicin V. This latter factor was also detected in isolates negative for the other virulence factors. On the basis of the type of erythrocytes agglutinated, we established 14 mannose-resistant hemagglutination patterns among the 37 strains tested, including 22 serum-resistant and/or aerobactin producing strains and 15 strains negative for these two characters. None of the strains produced alpha hemolysin, whereas two strains produced beta hemolysin and enterohemolysin, respectively. Congo red fixation was observed in 25 strains. No relationship could be detected between Congo red fixation and the presence of other virulence markers, such as serum resistance and aerobactin production. This study shows that among isolates originating from the feces of diarrheic chickens, the proportion of potentially diarrheagenic E. coli strains is low.

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called 'attaching and effacing' lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eae gamma-tir alpha-espA alpha-espB alpha (65%), eae beta-tir beta-espA beta-espB beta (29%), eae alpha-tir alpha-espA alpha-espB alpha (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.

Escherichia coli isolated from 204 cases of porcine postweaning diarrhoea were tested by PCR for the genes of cytotoxic necrotic factors (CNF) and of cytolethal dystending toxin (CDT). Selected strains were also examined by PCR for the presence of papC-, sfa-, f17-, f18-, and afa-specific sequences encoding P, S, F17, F18 fimbriae and afimbrial adhesins. A 5.9% (12/204) of the strains had cnf1 gene, and two of them had cdt gene as well. Further six cdt+ strains were detected which were cnf-negative. Most of the cnf1+ strains belonged to serogroups O2, O6, O8, O54 characteristic of necrotoxic E. coli (NTEC) of humans. All the cnf1+ strains possessed the genes for P or S fimbriae or both, but were negative for F4, F17, or F18 or afimbrial adhesins. Results suggest that these enteric isolates may have entero- and/or uropathogenic significance in weaned pigs, and may have zoonotic potential for humans.

Sixty-six rabbits, with no history of vaccination against myxomatosis and which had died of pulmonary lesions, were submitted for virological and serological tests for Myxoma virus (MV) infection and for bacteriological examinations. At post mortem, the diagnoses based on observed lesions were as follows: acute haemorrhagic pneumonia (38%); acute suppurative bronchopneumonia (35%); and fibrinohaemorrhagic bronchopneumonia with fibrinous pleuritis (27%). MV was isolated from 10% of the rabbits, mainly from those with acute haemorrhagic pneumonia. Serological evidence of MV infection was demonstrated in 44% of rabbits. Pathogenic bacteria species isolated from lungs were Pasteurella (spp. and multocida), Escherichia coli, Bordetella bronchiseptica and Pseudomonas aeruginosa, respectively, from 41, 11, 7 and 6% of samples. No relationship could be established between the presence of specific antibodies to MV and the observed pulmonary lesions or the results of the bacteriological examinations. A significant trend was established between the severity of the lesions and the results of the bacteriological examinations., (Copyright 2000 Harcourt Publishers Ltd.)

Two types of myxomatosis vaccine are available commercially, namely, vaccine prepared from the Shope fibroma virus (SFV) and that prepared from an attenuated myxoma virus (MV) strain, e.gSG33. An experiment was designed to compare two vaccination schemes for their ability to protect rabbits against challenge with either a virulent amyxomatous MV strain or a virulent nodular MV strain. Apart from a difference in the cutaneous expression of the disease, the two challenge strains resembled each other in respect of mortality rate, naso-conjunctival shedding of virus, and tissue infection. Vaccination with SFV alone failed to prevent clinical signs, naso-conjunctival shedding or tissue infection. Vaccination with SFV followed by a booster inoculation with SG33 protected rabbits against the development of clinical signs and significantly reduced both viral shedding in naso-conjunctival exudates and viral infection of eyelids, lungs and testes; virus was, however, isolated from testes of some surviving animals.

The virulence of five amyxomatous myxoma virus (MV) strains, the clinical and pathogenetic effects of which had been studied previously in specific pathogen-free (SPF) rabbits, was determined by inoculation of five groups of 10 crossbred New Zealand White/Californian conventional rabbits. A much more acute myxomatosis syndrome was produced in conventional rabbits than that reproduced previously in SPF animals. However, the main clinical signs were of the respiratory type. The MV strains MYX 254/95 and 801 appeared very virulent, killing all the inoculated animals. The strains MYX 217/95, MYX 555/94 and Saint Benoist were somewhat attenuated, killing only seven, six and six rabbits, respectively. Extensive lung lesions due to supervening bacterial infections were observed in 36 of the 39 rabbits that died. Lethality was found to be a better estimate of virulence than mean survival time. By 98 days after viral inoculation, all the surviving animals had completely recovered. At that time, they were immunosuppressed by treatment with adrenocorticotrophic hormone (ACTH) for 10 days to determine whether they still harboured the virus. After the ACTH treatment, eight of the 11 surviving rabbits showed clinical signs that resembled amyxomatous myxomatosis. All the virological examinations performed on naso-conjunctival exudate, on mononuclear cells, on eyelids and on ovaries remained negative but infectious virus was isolated from the testes of three of six surviving male rabbits.

Necrotoxigenic Escherichia coli (NTEC) isolated from animals and humans can belong to the same serogroups/types and produce or carry the genes coding for fimbrial and afimbrial adhesins of the same family, P, S, F17, and/or AFA, raising the question of a potential zoonotic source of human infection. The main purpose of this study was to compare 239 NTEC1 strains (45 from cattle, 65 from humans and 129 from piglets) and 98 NTEC2 strains from cattle, using a uniform and standardized typing scheme. The O serogroups and the biotypes recognized amongst NTEC1 and NTEC2 strains were quite varied, although some were more frequently observed (serogroups O2, O4, O6, O8, O18, O78, and O83 and biotypes 1, 2, 5, 6, and 9). Hybridization, results with gene probes for the P family (PAP probe), S family (SFA probe), AFA family (AFA probe), F17 family (F17 probe) of fimbrial and afimbrial adhesins, could differentiate most NTEC1 strains, which are PAP-, SFA- and/or AFA-positive, from NTEC2 strains, which are mainly F17- and/or AFA-positive, but were of no help in differentiating between NTEC1 strains from cattle, humans, and piglets. All but seven (98%) NTEC1 and NTEC2 strains were serum resistant, 199 (59%) produced an aerobactin, and colicin (I, V, or unidentified) was produced by 22-34% of them. On the other hand, more than 90% of the NTEC1 strains were haemolytic on sheep blood agar compared with only 40% of the NTEC2 strains. Production of a classical haemolysin, active on sheep erythrocytes, and hybridization with the PAP probe were associated in a majority of NTEC1 strains (63-81%), but very rarely in NTEC2 strains (3%). Production of enterohaemolysin and hybridization with the PAP probe were much less frequently associated in NTEC strains (1-9%). It was thus possible neither to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotizing Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additionnal methods.

Attaching and effacing Escherichia coli (AEEC) virulence genes include the eae, the tir, the espA and the espB genes. These genes have been sequenced from several AEEC strains. The sequences alignments revealed the presence of constant and variable regions. Multiplex polymerase chain reactions were developed, in order to determine the subtype of each gene present in a particular isolate. AEEC strains isolated from calves dead of diarrhea, 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 AEEC. Although, the human EHEC strains from serotype O157 possessed their own pathotype.

A monoclonal antibody (MAb) was obtained from a mouse immunized with solubilized outer membrane proteins extracted from a bovine enterohemorrhagic strain of Escherichia coli (EHEC), O26. The MAb produced a strong immunoblot reaction at approximately 21 kDa for an O26 strain containing the intimin gene (eae) and verocytotoxin (VT), but not with an O26 eae- and VT-negative strain, or O157 eae- and VT-positive strains. The MAb was used in a sandwich enzyme-linked immunosorbent assay (ELISA) format to screen strains from animal and human sources, and all reactive strains were characterized for the presence of eae and the gene encoding VT factors by PCR. The antigen was detected in a group of strains containing a high proportion of O26, the majority of which were eae positive with or without VT; these were isolated mostly from animal enteritis cases but included a small number of human enteric isolates. Nonreactors included eae-positive (with or without VT) O157 strains and one O26 strain. In a survey of mixed cultures from both animal and human enteric disease, ELISA-positive reactions were obtained from 7.1 to 11.2% of samples from bovine, porcine, ovine, and human sources. The two human O8 and ten animal O26 ELISA-reactive pure strains obtained from these samples contained six eae- and/or VT-positive strains; the other six strains lost their ELISA positivity following storage at -70 degrees C, after which none were found to contain either eae or VT factors. The association of the antigen detected by the MAb with significant enteropathogenic E. coli and EHEC virulence factors in isolates from both animal and human enteric infections indicates a diagnostic potential for the assay developed.

Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli isolated from cattle were studied by DNA colony hybridization to subtype their intimin-encoding (eae) gene with probes derived from the variable parts of the eae alpha gene of the human EPEC strain E2348/69, the eae gamma gene of the human O157:H7 EHEC strain ATCC43888, and the eae beta gene of the bovine O26:H- EHEC strain 193, whose eae gene was first cloned and sequenced during this work. The EPEC and EHEC had been isolated from diarrhoeic calves (143 EPEC and 48 EHEC) and from healthy animals at the slaughterhouse (10 EPEC and 34 EHEC). The 191 bovine EPEC and EHEC isolated from diseased calves were positive with the Eae beta probe (55 and 27% respectively) and with the Eae gamma probe (9 and 73% respectively), whereas 52 EPEC (36%) were negative with the Eae alpha, Eae beta, and Eae gamma probes. The results were different for the 44 bovine EPEC and EHEC isolated from healthy cattle at slaughterhouses: most tested positive with the Eae gamma probe (80 and 82% respectively) and the remaining (20 and 18% respectively) with the Eae beta probe. Nine O26 human EHEC tested positive with the Eae beta probe and seven O111 with the Eae gamma probe. The bovine and human EPEC and EHEC belonging to these two serogroups gave identical results: the 18 bovine and human O26 isolates tested positive with the Eae beta probe, whereas the 13 O111 isolates were positive with the Eae gamma probe. In contrast, the isolates belonging to other serogroups (O5, O15, O18, O20, and O118) gave more variable results. The eae beta and eae gamma, but not the eae alpha, variants were thus distributed amongst bovine EPEC and EHEC. The eae beta variant seemed to be more frequently associated with the presence of clinical signs in calves, but one third of EPEC from diarrhoeic calves carried an eae gene variant other than the alpha, beta, or gamma variants. In addition, the use of these gene probes did not enable differentiation between bovine and human EHEC belonging to the same O serogroup.

Vero/Shiga toxins (VT/Stx) have an A-B structure: the A subunit carries the enzymatic activity and the B subunit binds the toxin to the membrane receptor (Gb3 or Gb4). The VT/Stx inhibit protein synthesis in the target eucaryotic cells, mainly the endothelial cells of blood vessels. The VT/Stx are subdivided into two families. VT1/Stx1 is a homogeneous family of toxins identical to the Stx of Shigella dysenteriae. VT2/Stx2 is a more heterogeneous family of toxins more distantly related to this Stx toxin. The VT2/Stx2 variants can be distinguished by the polymerase chain reaction (PCR) and/or the reaction with monoclonal antibodies. The VT/Stx-producing Escherichia coli are also subdivided into two main groups on the basis of the presence or absence of additional properties: the enterohaemorrhagic E. coli (EHEC) induce the formation of attaching/effacing lesions and carry a 60 MD plasmid encoding a specific haemolysin (the enterohaemolysin); the vero/shiga-toxigenic E. coli (VTEC/STEC) do not show these properties. The EHEC are isolated from humans and ruminants, especially young calves. They are associated with haemorrhagic enterocolitis and its sequelae in humans, the haemolytic-uraemic syndrome (HUS). The VT/Stx play a role in the occurrence of blood in the faeces and in the HUS by their action on the endothelial cells of blood vessels in the intestinal submucosa and in the renal glomeruli, after resorption through the intestinal walls. The VTEC/STEC are isolated from piglets, calves and humans. In recently weaned piglets, they cause the oedema disease, an enterotoxaemia characterized by subcutaneous, mesenteric and cerebral oedemas, with nervous disorders as main clinical signs. The oedema disease is the consequence of the action of the VT/Stx on the endothelial cells of blood vessels in various organs. In calves and humans, the role in disease of VTEC/STEC is controversial, but they could be associated with some cases of diarrhoea and HUS. The case of the O157:H7 EHEC which are present in healthy cattle of various ages, but are highly virulent for humans is of special interest. The potential zoonotic aspect of VT/Stx-producing E. coli infections in animals is detailed chapter by chapter. Prophylaxis of these infections by vaccination is the subject of the discussion on the future of the research studies on these pathogenic bacteria.

Enteropathogenic Escherichia coli (EPEC) produce attaching and effacing lesions. The genes responsible for this lesion are clustered on the chromosome forming a 35.5 kilobase pathogenesis island called LEE. The LEE was identified, characterized and completely sequenced from the human EPEC strain E2348/69. The LEE carries genes coding for: a type III secretion system (genes esc and sep), the translocated intimin receptor (gene tir), the outer membrane protein intimin (gene eae) and the E. coli secreted proteins EspA, EspB, and EspD (genes esp). In addition to man and farm animals, EPEC are also isolated from dogs and cats. We studied structurally and functionally the LEE of dog and cat EPEC. First, we used four probes scattered along the LEE to identify the presence of a LEE in canine and feline EPEC isolates. Second, by PCR, we checked the presence of genes homologous to eae, sep, esp, and tir genes in these strains. Third, since the four types of eae and tir genes were described, we developed a multiplex PCR in order to determine the type of eae and tir genes present in each strain. Fourth, we determined by PCR the site of the LEE insertion on the chromosome. Fifth, we tested several of the canine EPEC in their capacity to induce attaching and effacing lesions in the rabbit intestinal loop assay. We can conclude from this study: first, that the a LEE-like structure is present in all tested strains and that it contains genes homologous to esp, sep, tir, and eae genes; second, that there is some preferential associations between the type of eae gene and the type of tir gene present in a strain; third, that the majority of the tested strains contained a LEE located elsewhere on the chromosome in comparison to the human EPEC strain E2348/69; and fourth that dog EPEC were able to induce attaching and effacing lesions in rabbit ileal loop assay.

CNF2-producing necrotoxigenic E. coli (NTEC2) are associated with diarrhoea and septicaemia in calves. We orally inoculated neonatal calves with a NTEC2 strain in order to reproduce clinical signs and lesions. We observed diarrhoea in each inoculated calf, bacteraemia (80%), the presence of CNF2+ bacteria in the lungs (80%) and in the liver (20%). The observed lesions were inflammation of the entire gut, hypertrophy of the mesenteric lymph nodes and hepatisation of the lungs. We were unable to detect characteristic lesions that are classical signs of septicaemia.

Attaching and effacing rabbit enteropathogenic Escherichia coli (REPEC) of the O103 serogroup adhere diffusely on HeLa cells and trigger a slow progressive cytopathic effect (CPE) characterized by the recruitment of vinculin and the assembly of actin stress fibres. In contrast to REPEC O103, the reference human EPEC strain E2348/69 is unable to trigger the CPE. In this study, we have shown first that the fimbrial adhesin AF/R2, which mediates the diffuse adhesion of REPEC O103, was not sufficient to induce the CPE capability upon E2348/69. Non-polar mutants of REPEC O103 for espA, espB, espD and eae were then constructed. The four mutants were unable to induce attaching and effacing lesions in the rabbit ileal loop model. The esp mutants were no longer able to induce the CPE, whereas the eae mutant still induced the CPE. Each espA, -B, -D mutant could be fully complemented in trans by the corresponding cloned esp genes from both the parental strain and the CPE-negative E2348/69 strain, indicating that no single esp encodes the information needed to confer the CPE phenotype. In conclusion, the CPE is the first example of an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton by certain EPEC strains.

Bovine enterotoxaemia is an acute to peracute syndrome occurring mainly in calves and characterized by the sudden or very rapid death of the calf, with colics, convulsions and nervous disorders as clinical signs, if any. The most pronounced lesion is a necrohaemorrhagic enteritis of the jejunum, the ileum, and sometimes the colon. Suckling beef calves are the most frequently affected ones. In 67% of the 78 field cases investigated, some kind of stress was observed 24 to 36 hours prior to the death: change in diet or pasture, vaccination... The most frequently isolated bacteria, and the one isolated in highest numbers, was non-sporulated non-enterotoxigenic toxinotype A Clostridium perfringens. Reproduction of the lesions was successful in a ligated intestinal loop assay in one calf with a few of these strains, more especially with one of them, which was shown later to produce another recently described toxin, the beta 2 toxin. A role for this beta 2 toxin in bovine enterotoxaemia is thus speculated for future research.

Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves. The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E. coli strain E2348/69 in which the virulence correlated with the presence of a 35.4 kb pathogenesis island called LEE. We showed that several strains isolated from calves with diarrhea were able to produce attaching and effacing lesions in a rabbit ileal loop model and that they possess a pathogenesis island related to the LEE. Moreover, we showed that the LEE from bovine strains was inserted mainly at a different position in the chromosome compared to the human enteropathogenic E. coli strain E2348/69.