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Avian pathogenic Escherichia coli

Professor Jacques Mainil, University of Leige, Belgium Introduction Strictly speaking avian pathogenic Escherichia coli (APEC) are E. coli strain that can cause disease in birds of various ages : septicaemia, chronic respiratory disease, vitellus infection, salpyngitidis, peritonitis, chronic skin infections, osteomyelitis, swollen head syndrome, Today nevertheless, APEC represent E. coli strains causing invasive infection of poultry (chickens and turkeys), more especially of broilers, with the respiratory tract as portal of entry : septicaemia, peritonitis, perihepatitis, air sacculitis, osteomyelitis, This presentation will therefore focus on the pathogenesis and virulence properties of these particular invasive strains. Purpose of the presentation The pathogenesis of APEC infections comprises four stages, just like any infection caused by invasive E. coli strains in mammals : colonisation of the respiratory tract, crossing of the epithelium and penetration into the mucosa of the respiratory organs (air sacs), survival and multiplication in the blood stream and in the internal organs, production of deleterious effects on the eukaryotic cells and tissues leading to lesions followed by clinical signs. Amazingly enough we know very little of the specific virulence properties of invasive E. coli strains, including APEC, compared to diarrhoeagenic E. coli strains. This lack of knowledge represents a problem in the diagnosis and epidemiology of E. coli strains isolated from poultry and prompted Dr Maryvonne Moulin, from the INRA Tours, to coordinate a European Shared Cost project (FAIR6-CT98-4093) whose purpose was to (i) identify new putative virulence-associated properties of APEC ; (ii) study the role of some of these new properties in their pathogenesis; and (iii) determine their sensitivity and resistance to antibiotics classically used in poultry farming. The five participating teams bio/serotyped a collection of 1600 strains isolated in Belgium, France and Spain, from blood or internal organs of chickens, turkeys and ducks, and studied the prevalence of genes coding for different adhesins, toxins, iron chelators and survival properties. The ultimate purpose was (i) the development of a more precise and complete diagnosis and typing scheme than the current one essentially based upon partial serotyping ; and (ii) the identification of new effectors that could be included in poultry vaccines to improve their efficacy. Colonisation of the respiratory tract. Like almost all E. coli strains, APEC produce F1 fimbriae that are expressed in the trachea, lungs and aerial sacs. Though once considered as playing an important role in the colonisation of the respiratory tract, F1 fimbriae are no more regarded as a virulence factor of APEC. Actually specific colonisation factors and

adhesins of APEC are still a matter of debate. Until recently only fimbrial adhesins belonging to the so-called P family (of the F11 serotype) had been identified on APEC. Still only 20 to 25% of strains are positive for P fimbriae and their role in the colonisation of the respiratory tract has not been firmly proved. During the FAIR6-CT98-4093 shared cost project genes coding for F17 and S fimbriae and for Afa-VIII adhesins were identified by colony hybridisation and by PCR, each in ca. 5% of the APEC, sometimes in combination with each other or with genes coding for P fimbriae (Grardin et al., 2000 ; Mellata et al., 2000 ; Stordeur et al., 2002 and 2003). Their actual role in the colonisation of the respiratory tract, or in another step of the pathogenesis of APEC, must still be studied. A thermo-sensitive haemaglutinin (Tsh) is preferentially associated with APEC and very rarely found on E. coli strains isolated from faeces of healthy chickens : 95% of Tsh-positive avian E. coli are APEC. Tsh may play a role in the colonisation of the respiratory epithelium (Dozois et al., 2000). Crossing of the epithelia and mucosae. The mechanisms by which the APEC cross the respiratory epithelia and penetrate deeply into the mucosa and sub-mucosa are still unidentified to my knowledge. Invasion of the blood stream and internal organs. Once they reach the blood stream, APEC face several dangers : opsonisation by the C3b component of the complement, bactericidal activity of the membrane attack complex (MAC) formed by the C5b, C6, C7, C8 and C9 components of the complement and phagocytosis by neutrophils and macrophages. E. coli are by essence no intracellular bacteria. Once inside the phagocytes they can not survive. The only defensive strategy is not to enter into the phagocytes. The presence of certain O and K antigens at the surface of the bacteria helps APEC to defend themselves by avoiding opsonisation by C3b and phagocytosis. It is common knowledge for instance that many APEC belong to the O1, O2 or O78 somatic serogroups that probably interfere with the deposit of C3b at the bacterial cell surface. The K1 antigen, containing N-acetyl neuraminidic acid, also negatively interacts with C3b. Moreover the K1 antigen is poorly immunogenic. Similarly the same bacterial cell surface components hamper the deposit of the MAC at the cell surface and its incorporation into the outer membrane, saving the APEC from lysis and death. It is believed that these antigens prevent the deposition of C3b and of MAC, at least in part, by steric hindrance. Moreover more than 80% of APEC produce particular proteins (TraT, Iss) that incorporate inside the outer membrane and prevent the deposition of the MAC by a still unidentified mechanism. Anyhow thanks to the role played by these different mechanisms the APEC survive the host defences. But there is another obstacle to the survival and multiplication of invasive bacteria : the very low concentration of free iron (about 10-18 mol/L). Iron is indeed chelated by host siderophores either in the extracellular fluids (transferrin, lactoferrin, ovotransferrin) or intra-cellularly (ferritin) as a defence reaction in front of the bacterial aggression. Bacteria must find iron if they want to survive and

multiply. Several mechanisms have been described amongst various pathogens but the most frequent one of invasive E. coli is the production of siderophores to take the iron back from the host siderophores. The E. coli most powerful siderophores are enterobactin, aerobactin and yersiniabactin. Enterobactin is produced by most E. coli strains but aerobactin is produced more especially by invasive E. coli. The aerobactin is advantageous for pathogenic bacteria compared to the enterobactin because it is recycled after delivery of the iron atom to the bacterial cell and it is still active during the inflammation process. Aerobactin-encoding genes are present in more than 80% of APEC. Yersiniabactin is a more recently described siderophore in E. coli and its prevalence amongst APEC has not been studied to my knowledge. Another mechanism is the production of haemolysins which cause the lysis of erythrocytes and the liberation of iron from haemoglobin. But very few APEC are positive for the production of haemolysins. Toxicity. Though production of different cytotoxins by a few APEC ( -haemolysin, cytotoxic necrotising factor 1, cytolethal distending toxins, shiga toxins, E. coli vacuolating factor) has been demonstrated in some studies, it is still not clear today whether the cell and tissue damages caused during APEC infection are the results of the action of still unidentified toxins or more simply of the inflammatory response of the host. Conclusions. In summary we still have a lot to learn about the virulence factors and properties of avian invasive E. coli strains forming the group of so-called APEC strains. But gain of this knowledge is very important, not to say indispensable to (i) design a more complete and more precise diagnosis and typing scheme of E. coli isolated from internal organs of poultry ; (ii) to identify the actual source of infection within a herd ; (iii) design more efficient vaccines than the one(s) currently available thanks to the recognition of the heterogeneity of APEC strains. Several molecular, bacterial and animal studies must still be accomplished if we want to reach these objectives. Identification of new adhesins for instance may also lead to the recognition of poultry lines which are resistant to colibacillosis, understanding of the genetic background of resistance to colonisation and development of an E. coli-resistant poultry farming system. But we are far from there yet. Review manuscripts - Dho-Moulin M., Fairbrother J.M. Avian pathogenic Escherichia coli. Vet. Res., 1999, 30, 299-316. - Stordeur P., Mainil J. La colibacillose aviaire. Ann. Md. Vt., 2002, 146, 11-18. - Vandekerckhvove D., De Herdt P., Laevens H., Pasmans F. Colibacillosis bij de kip. Vlaams Diergeneeskd. Tijdschr., 2003, 72, 180-190.

Research manuscripts - Dozois C., Dho-Moulin M., Bre A., Fairbrother J.M., Desautels C., Curtiss III R. Relationship between the Tsh autotransporter and pathogenicity of avian Escherichia coli and localization and anaysis of the tsh genetic region. Infect. Immun., 2000, 68, 4145-4154. - Grardin J., Lalioui L., Jacquemin E., Le Bougunec C., Mainil J. The afarelated gene cluster in necrotoxigenic and other Escherichia coli from animals belongs to the afa-8 variant. Vet. Microbiol., 2000, 76, 1-10. - Mellata M., Bakour R. Jacquemin E., Mainil J. Genotypical and phenotypical characterization of potential virulence of intestinal avian Escherichia coli strains isolated in Algeria. Avian Dis., 2001, 45, 670-679. - Stordeur P., Marlier D., Blanco J., Oswald E., Biet F., Dho-Moulin M., Mainil J. Examination of Escherichia coli from poultry for selected adhesion genes important in disease caused by mammalian pathogenic E. coli. Vet. Microbiol., 2002, 84, 231-241. - Stordeur P., Beaupain N., Mainil J. Caractrisation gnotypique de souches invasives aviaires dEscherichia coli isoles en Belgique. Ann. Md. Vt., 2003, 147, 275-280.