Avian Pathology 15503079457 (Print 1465-3338 Online Jounal homepage htos//ynestandontnecomvolcovp20 Colibacillosis in poultry: unravelling the molecular basis of virulence of avian pathogenic Escherichia coli in their natural hosts Francis Dziva & Mark P. Stevens To cite this article: Francis Dziva & Mark P. Stevens (2008) Colibacillosis in poultry: unravelling the molecular basis of virulence of avian pathogenic Escherichia colin their natural hosts, Avian Pathology, 37:4, 355-366, DOI: 10,1080/03079450802216652 To link to this article: https://doi.org/10.1080/03079450802216652 CF suomityourarie ois ural & Lo arc views: 12168 CB) cing articles: 55 view cing articles Full Terms & Conditions of access and use can be found at bhtepsu/www.tandfoniine.com/action/journalinformation2journalCode=cavp20Taylor Francis Avian Pathology (August 2008) 3(4), 355-366 REVIEW ARTICLE Colibacillosis in poultry: unravelling the molecular basis of virulence of avian pathogenic Escherichia coli in their natural hosts Francis Dziva* and Mark P. Stevens Division of Microbiology, Instiute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK Avian colibacillosis is caused by a group of pathogens designated avian pathogenic Escherichia coli (APEC). Despite being known for over a century, avian colibacillosis remains one of the major endemic diseases afflicting the poultry industry worldwide. Autologous bacterins provide limited serotype-specific protection, yet multiple serogroups are associated with disease, especially Ol, 02 and O78 among many others. Experimental infection models have facilitated the identification of some key APEC virulence genes and have allowed testing of vaccine candidates. Well-recognized virulence factors include Type | (F1) and P (Pap! Prs) fimbriae for colonization, IbeA for invasion, iron acquisition systems, Tra and Iss for serum survival, K and © antigens for anti-phagocytic activity, and a temperature-sensitive haemagglutinin of imprecise function. Intriguingly, these factors do not occur universally among APEC, suggesting the presence of multiple alternative mechanisms mediating pathogen bility of the first complete APEC ‘genome sequence ean be expected to accelerate the identification of bacterial genes expressed during infection and required for virulence. High-throughput molecular approaches like signature-tagged transposon mutagenesis have already proved invaluable in revealing portfolios of genes expressed by pathogenic bacteria during infection, and this has enabled identification of APEC 02 factors required for septicaemia in the chicken model. Complimentary approaches, such as in vito-induced antigen technology, exist to define the activities of APEC or riro. In recent years, reverse vaccinology and immuno-proteomic ‘approaches have also enabled identification of novel vaccine candidates in other bacterial pathogens. Collectively, such information provides the basis for the development or improvement of strategies to control APEC infections in the food-producing avian species. Introduction ‘The association of Escherichia coli strains with disease conditions in avian species was recognized over a century ‘ago (cited by Sojka & Camaghan, 1961), but these coligranuloma, omphlitis, cellulitis and osteomyelitis! arthritis may be encountered (reviewed in Barnes & Gross, 1997), In. some instances, APEC has been Sinains were never accorded a special status, Today, E. coli strains causing systemic disease in poultry (avian, colibacillosis) are termed avian pathogenic E. col! {APEQ). Colibacillosis is a disease of severe economic significance to all poultry producers worldwide and is characterized by a diverse array of lesions. Recent reports in Western Europe implicate @ resurgence of this disease in the poultry industry, particularly in chicken layers (Zanella er al., 2000; Vandekerchove et al, 2004; Jordan et al, 2005). Depending on the virulence status of the strain, host status and presence and type of predisposing factors, the infection manifests ‘as.an initial septicaemia that is followed by either sudden, ‘death oF localized inflammation in multiple organs, The most common lesions associated with colibacillosis are perihepatitis, airsacculitis and pericarditis, although ‘other syndromes such as ege peritonitis, salp ‘associated with peculiar diseases in specie avian species, In chickens, swollen head syndrome often results from synergistic infection of turkey thinotracheitis virus and E.coli (Morley & Thompson, 1984; Picaulte” al., 1987; Stehling et ai, 2003). Turkey rhinotracheits virus is believed to cause initial acute rhinitis that is followed by invasion of the facial subcutaneous tissues by coli (Picault er al, 1987). In turkeys, APEC also causes osteomyelitis complex characterized by lesions including, agrven discolouration of the liver, athritis'synovitis, soft- tissue abscesses, and osteomyelitis of the proximal tibia in an otherwise normal-appearing processed turkey careass (Hull et af, 2000), Typically, this disease is observed in male adolescent turkeys that have low levels of cell-mediated immunity (Bayyari ef al, 1997; Hu et al, 2000), implying that defects in the immunity of individual male turkeys lead to disease. “To whom correspondence shuld be akrowed. Te: 44 1635 STR SI Fax 441635577255. Ema ransriaabre ac ok Resist 25 March 208 ISSN 0379 DOH. 1080 0509440221642 rin)ISSN 146-3338 (onlin 084035512 4 200 Houphon Trust Lad356. F, Driva and M. P, Stevens ‘The natural route of infection for APEC is not clearly dlfined, although the oral and respiratory routes soem 10 be significant modes of entry. An estimated 10% to 15% ‘of avian alimentary tract coliforms have been reported t0 belong to potentially pathogenic APEC serotypes (Harry & Hemsley, 1965). Consistently, both virulent and avirulent E: coff have been shown to colonize and persist elfciently in the intestinal tract, with extra-intestinal translocation occurring only in the presence of stressors (Dominick & Jensen, 1984; Leitner & Heller, 1992). The implications of the presence of both pathogenic and commensal strains in the intestinal eavironment are not clearly discernible, although it ean speculated that this could be a leading source of a diverse range of APEC strains as reported for atypical enterohaemorthagic E coli and enteropathogenic E.coli (Homnitzky et al 2005). Strikingly, the intestinal location of APEC provides an excellent opportunity for dissemination into the envionment and transmission via faeces. APEC has been reported to persist in the dry environ- ment, and dustin poultry houses may harbour up to 10° colony-forming units of E. coli per gram (Harty, 1964). Inhalation of this contaminated dust is believed to lead lo systemic APEC infections. Infection of eges may ‘occur at laying or during formation in the oviduet, often leading to embryo and carly chick mortality. Inthe United Kingdom, salpingo-peritonitis was the most common form of avian colibacillosis observed in laying hens (lordan er a, 2005), but what influences presenta- tion of this pathology is unknown. ‘A major predisposing factor for systemic APEC infections is stress, which can be induced by a variety ‘of agents or inappropriate husbandry practices (see Barnes & Gross, 1997). There are numerous other predisposing factors associated with APEC infections (Barnes & Gross, 197) and these have led to the notion that this disease could result from opportunistic infec- tion, thus underestimating the virulence of APEC. Consistent with this belief, many serotypes of E.coli including those believed to be harmlessly carried in the imestinal tract have becn recovered from diseased birds (Blanco er al., 1998; MePeake et al., 2005; Rodriguez Sick et a, 20054). Indeed, earlier “workers regarded systemic Ecol infoction as an indicator of physiological sress in chickens (Kendler & Harry, 1967). Furthermore, experimental evidence indicates. that extraintestinal translocation occurs only in the presence of stressors (Leitner & Heller, 1992). This ability to translocate across either the intestinal or respiratory mucosae and disseminate systemically makes APEC also belong to the extra-intestinal pathogen £. coli (exPEC) group. The exPEC group includes neonatal-causing meningitis E coli (NMEC) and uropathogenic £ coli (UPEC), which ‘predominantly translocate from the intestinal tract ‘Our current understanding of the pathogenesis of colibacillosis has been enhanced by the availability of experimental infection models in target hosts. A con- siderable amount of information on avian colibacillosis, row exists and the molecular virulence determinants of APEC are slowly being unravelled, The recent avail- ability of an APEC OL:KI:H7 complete genome se- {quence (Johnson e¢ al, 20074) provides a turning point in our understanding of APEC and also an appropriate launching pad for the application of genetic methods to define the APEC puthotype and virulence, Novel ap- proaches to survey the repertoire, sequence, expression and role in virulence of APEC genes in controlled infection models provide an excellent opportunity to improve our knowledge on APEC virulence mechanisms. Here, we re-examine the APEC pathotype and progress towards unravelling APEC virulence factors mediating carriage and systemic translocation in their natural hosts, ‘The APEC pathotype Unlike other pathogenic groups of E. col, no single trait ‘or group of traits defines the APEC pathotype. Some of the phenotypic and genotypic charactersties associated with this group of pathogens are reviewed below, ‘and serotyping are often under- taken on isolates recovered Irom cases of colibacillosis. In most countries, O1, 02 and O78 represent major E, ‘oli serogroups isolated trom diseased birds (Sojka & Carnaghan, 1961: Cheville & Arp, 1978; Cloud et al, 1985; Whittam & Wilson, 1988; Dozois er al, 1992, Ewers et a, 2004). Consequently, representative strains from these serotypes provide the focus for unravelling APEC virulence mechanisms and for the development ‘and evaluation of vaccine candidates. These predomi- nant serogroups can also be recovered from faeces of apparently healthy birds (Blanco er al, 1998), reinfor- ting the notion that the intestinal tract may be an important natural reservoir for APEC and that predis- Posing factors may be required to produce disease. Significantly, several studies have reported shared char- acteristics including serogroups between commensal E, coli and APEC (Heller & Drabkin, 1977; Blanco er a, 1998; MePeake er al, 2005; Rodriguez-Sick er al, 2005a), implying that APEC could arise Irom commen- ‘al. E.coli following acquisition of pathogenic traits. Indeed, widespread variation in phenotypic and viru- lence characteristics within a single APEC serotype has been reported. Outer membrane protein profiles, vitu- lence and multilocus enzyme electrophoresis profiles within a serotype were some of observed. variable characteristics (Whitlam & Wikon, 1988: Kapur eal 1992; Chaffer et a., 1999). Despite the variable pheno type, tests for motility, haemolysis and lactose fermenta- tion are sometimes tested in conjunction with serotyping, colicin V and aerobactin production, com- plement resistance and embryo lethality tests to char- acterize APEC isolates Genotype. Putaive virulence gene surveys. Plasmi mediated horizontal gene transfer resulls in extensive diversity within the APEC. The APEC plasmids carry several putative virulence factors, although some of these have been encountered in E. col isolates from apparently healthy birds (MePeake er al., 2005; Rodriguez-Siek et al, 2005a; Kawano et al., 3006). A high degree of variability in the number, size and virulence traits cartied ‘on large plasmids exists in both APEC and isolates from apparently healthy birds (Doetkott et al, 1996). But a recent study (Johnson et al, 2007b) failed to detect APEC-specific plasmid replicons and colicin-related ‘genes in commensal strains, Undoubtedly, APEC plas- mids carry a significant number of virulence genes that contribute to the present definition of the APEC tgenolype. Notably, « 94 kb region of a 180 kb ColV. plasmid carries genes encoding for iron acquisition andtransport systems, and these are more prevalent in APEC than avian faccal Escherichia coli (AFEC) ohnson et al., 2006). Importantly, a link between APEC virulence and possession of Col¥ plasmids has been rewaled by several studies (Ginns er al, 2000; Johnson eal, 2002; Gibbs era, 2003; Tivendale er a. 2004). Transformation of an avirulent wild-type E. coli strain with a recombinant plasmid (pHKI1) encoding for colicin V led to inereased colonization of the chicken trachea (Wooley er ai, 1998). Recently, transferring 2 APEC plasmids (ColV and large R) into a commensal ‘oli strain by conjugation enhanced its abilities to Kill chicken embryos, grow in human urine and colonize the murine kidney (Skyberg er al, 2006). Based on a selection of common traits that include plasmid-borne traits, 1wo multiplex polymerase chain reaction (PCR) protocols have recently been described for the identifica- tion of APEC (Skyberg eal, 2003; Ewers er a, 2008) Strains from diseased birds were classified as APEC if they harboured at least four of the eight genes included in the study—P fimbriae (papC), aerobactin (iueD), iron repressible protein (irp2),temperature-senstive het slutinin (ish), vacuolating autotransporter protein (ea, centeroaggrezative toxin (ast), inereased serum survival protein (ss) and colicin plasmid operon genes (cvalei) Whilst non-pathogenic isolates (as evidenced by chicken infection studies) possessed either none or at most three ‘ofthe genes (Ewers etal, 2005) Recently, four multiplex PCRs have been developed for virulence genotyping and for establishing a phylogenetic link between avian and hhuman exPEC strains (Ewers ef a, 2007). Surprisingly, there is lack of detailed information on the combinations ‘of genes essential for inducing systemic APEC infections. Bul a previous study suggested that the combinations ‘TshvPil/luc, TshiPap/lue pathotypes and Tsh and uc Virulence-associated markers were important Factors for APEC (Ngeleka er al, 2002), A limited association between the virulence gene pattern and the serogroup of APEC strains exists (Ewers et al, 2004). Dillerent associations of virulence genes could reflect the existence of sbpatonpes orient natones wii the sent APEC group (Delicato et al, 2003), hence Uitfeent virulence mechanisms might be employed by the different putative subpathotypes. Clearly, the defini- tion of APEC merits further revision, Comparative genomics. Eforts to understand the genetic basis of APEC virulence have been facilitated by comparative genomic studies involving. strains from diseased birds with their counterparts from apparently healthy birds or laboratory-adapled strains, and also through molecular typing techniques. Genomic suppres- sion subtractive hybridization (GSSH) has enabled identification of several important APEC virulence factors absent in non-pathogenic E coli (Brown & Curtis III, 1996; Stock et a Schouler et al, 2008; Mokady et al, 2005; Kariyawasam er ai, 20060, 2007), Independent application of the same approach in different O2 APEC strains led to identification of specific but not identical sequences (Stocki er al, 2002: Schouler er al, 2004), suggesting the presence of a variety of potential virulence factors, These findings were unusual since a high degree of genetic diversity exists between APEC serogroups (Mokady etal, 2005) and as ‘well as within a serogroup (White ea, 1993a). Virulence determinants of APEC. 357 Evidence linking some APEC clones with human exPEC strains, particularly UPEC and NMEC, is being, uncovered, Comparative analyses between APEC and human exPEC have revealed striking similarities in genomic islands, virulence genes, overlapping O ser ‘ogroups and phylogeny (Stordeur et al., 2002; Schouler er al, 2004; Germon et al, 2005; Rodriguez-Siek et al, 200b; Ron, 2006; Chouikha et a, 2006; Johnson etal, 20074; Ewers et af 2007; Kariyawasam et al, 2007) Furthermore, earlier work had shown APEC strains to be easily transmitted to humans (Linton et al, 1977; Ojeniyi, 1989). Indeed, studies have shown that some APEC strains could belong to the same clones as human exPEC strains (Achtman er al, 1986; White er al, 1993), Recently, it has been reported that very closely related clones of serotype O18:K I:H7 could be recovered from extra-intestinal infections in humans and chickens and that isolates from both species were virulent for chicks (Moulin-Schouleur er af, 2006). Consistent with these observations, whole genome sequence analysis has revealed a high degree of similarity between APEC and exPEC, with only 4.5% of the APEC OK IH genome not found in three exPEC genomes (Johnson et al, 2007a). Furthermore, analysis of a diverse collection of APEC, UPEC and NMEC strains suggested that poultry ‘may serve as a vehicle or even reservoir for human exPEC strains and that APEC may be the source of virulence-associated genes for exPEC strains (Ewers et al, 2007), suggesting the possibilty ofa food-borne link between these pathogroups. Molecular typing methods have also been employed to study APEC, but none have revealed an APEC. c genotype. Extensive genetic diversity among APEC and within a serotype has been demonstrated by enterobacterial repetitive intergenic consensus-PCR typing (Carvalho de Moura et al, 2001) and random amplification of polymorphic DNA (Maurer et af, 1998). Predominant serotypes associated with colibacil- losis appear phylogenetically distant by multilocus. enzyme electrophoresis (White er al, 1993a), although pathogenic clones are relatively closely related. when compared with commensal strains (da Silveira et al 2003), Multilocus sequence typing of O78 strains has revealed closely related clones to reside in different hosts and a strong correlation between virulence and clonal origin (Adiri et al, 2003). PCR-based phylotyping and rmultitocus sequence typing have also revealed a. link between APEC and human exPEC (Moulin-Schouleur et al, 2007), further suggesting the potential food-borne source of human exPEC Progress towards unravelling APEC virulence factors nization factors. Early work established the respira tory tract to be a significant route of entry into the host (Gross, 1961), whilst the intestinal tract has been reported to be a reservoir for both pathogenic and non-pathogenic strains (Harry & Hemsley, 1965). Colo- ization of multiple internal organs occurs in birds that survive initial septicaemia. In this respect, expression of several adhesins by APEC could enhance preferential colonization of different sites during the infection process including intestinal carriage (Stordeur er al, 2002)358 F, Driva and M. P, Stevens Fimbrial adhesins. Fimbriae are proteinaceaus filaments ‘or appendages expressed on the bacterial surface that are believed to mediate adherence to host cells and these consist of different types. Mannose-binding type 1 (F1) fimbriae are commonly encountered in APEC, with Frequencies ranging between 70% and 100% (Janssen et ‘al, 2001; Dho-Moulin & Fairbrother, 1999) The role of these fimbriae in colonization has partly been confirmed using tracheal explants (La Ragione ef al, 2000a) and intestinal explants where they target follicleassociated epithelium (Edelman er af, 2003). Although expression (of these fimbriae has been demonstrated in eit follow= ing experimental infection (Dozois et al, 1994; Pour- bakhsh er af, 1997a,b), their role in virulence has remained questionable, A defined fim mutant colonized the trachea, lungs and internal organs of germ-free chickens to the same extent as the parent strtin (Mare et al, 1998), Conversely, a defined fimH mutant lacking, adherence properties in viiro on chicken pharyngeal and tracheal epithelial cells actually exhibited an increased adherence to tracheal mucosa in vivo (Arne etal 240) Besides wellcharacterized Type I fimbriae, P (Papi Prs) fimbrial adhesins have boon reported to be ex- pressed by bacteria that colonize internal organs but not the trachea (Pourbakhsh ef al, 19976) implying that they may be required for systemic translocation. How- ever, a survey by PCR revealed that 76% of the 1601 avian E, coli isolates lacked a pap gene cluster that encodes for these fimbriae (Stordeur et al, 2002). ‘Among the pup-negative strains were fI7-positive andi ‘oF afa-8-positive strains, adhesins commonly found in pathogenic E. coli of mammals (Bouguenee & Bertin, 1999), Strains harbouring the 17 gene cluster were lethal for I-day-old chicks and induced classical colibacillosis lesions indicating that P fimbriae may not essential for pathogenesis (Stordeur er al, 2004) and that their Function may be substituted by other adhesins. Several other types of Fimbriae have been reported in APEC. Avian E.coli 1 (AC/1) fimbriae of the S-fimbrial adhesin family (Bubai er al, 1997, 2000) have been reported to mediate adherence to avian epithelial tissues {nitro and in vivo (Yerushalmi er al., 1990). Type IV pili encoded on a conjugative plasmid pO78V have recently been described but their role in pathogenesis remains unclear (Gophna et af, 2003). Additionally, a number of putative adhesins were recently identified by selective capture of transcribed sequences (SCOTS) induced i rico (Dozois et a, 2003), including the recently named, Sig fimbriae (Lymberopoulos et af., 2006). Its now clear that Sig fimbriae resemble previously described long polar fimbriae in APEC; both are located between conserved genes ginS and. pstS and contribute 10 adherence to epithelial cells (Ideses er af, 200Sa:, Lymberopoulos et all, 2006). These fimbriae enable adherence of a non-fimbriated E. coli K-12 strain 10, avian lung sections and contribute to colonization of airsacs but not lungs and trachea (Lymberopoulos er al., 2006). ‘Other pili-related factors identified by SCOTS include ‘iI and pitQ, which are believed to encode for plasmid- bore type IV pili. Recently, an £. colt common pilus that oceurs at high frequencies in most pathogenic E. coli, including APEC, has been reported to be a universal adhesin mediating epithelial cell colonization (Rendén et al, 2007), Afimbrial adhesins. Although commonly associated with pathogenic E.coli strains from mammals, the afa-S gene Cluster has been detected amongst several of the pap- negative avian strains (Stordeur et al., 2002). The afa-8 ‘operon encodes for an afimbrial adhesin that contributes, to virulence for I-day-old chicks and induction of classical colbacillosi in a manner similar to /17-positive ‘or pap-positive strains (Stordeur eral, 2004). In addition, to this well-characterized adhesin, several other putative colonization factors have been described. Curl are coiled Surface structures found in most £. coli strains whose role in colonization has been deduced from their ability to bind fibronectin (Olsén et al., 1989), to adhere to chick explant tissues (La Ragione er af. 2000a) and theit contribution to persistence in I-day-old chicks (La Ragione et al., 20005). An autotransporter temper ture-sensitve haemagglutinin (Tsh) has been suggested to contribute to early. stages of infection including colonization of airsaes but not subsequent generalized infection (Dozois er al., 2000). Consistent with these ‘observations, Tsh can adhere to red blood cals and also ind to extracellular’ matrix proteins (Kostakioti_ & Staphopoulos, 2004). Tsh is produced as a 140 kDa protein that is processed into a 33 kDa agglutinin fragment and a 106 kDa fragment with mucinase activity (Kobayashi et al, 2007). Interestingly, the role of ‘autotransporter proteins in colonization and adherence to epithelial cells has previously been suggested (Het dderson ef a, 2004), and recently we have shown EspP, ‘an_autotransporter protein of E. coli O1ST:HT, to influence intestinal colonization of calves (Dziva et al. 2007). Intimin, an adhesin associated with selected diarrheagenic E coli, was doteoted only ina few avian intestinal E. coli strains (Stordeur et a, 2002). Other putative afimbrial adhesins identified by SCOTS include TefD and a ColV plasmid-encoded haemoglobin pro- tease (Dozois et al, 2003), but their relevance to colonization remains to be confirmed. Invasive factors. It remains unclear where and how APEC reaches the bloodstream. Avian airsacs lack resident defence mechanisms and rely on recruited inflammatory heterophils followed by macrophages (Ficken & Barnes, 1989). Jn veo experiments have shown, that APEC are able to survive macrophage activity (Pourbakhsh e7 al, 19974; Mellata er al, 20036), Whether bacteria gain entry into the bloodstream following upiake by macrophages or there is a direct invasion after damage to the airsae or lung epithelia is unknown, Entry through pulmonary lymphatics has bbeen suggested (Cheville & Arp, 1978) and bacteria have been shown to be abundant in the blood as early as 3 h following intra-airsae inoculation (Piercy & West, 1976; Pourbakhsh er al, 1997; Stordeur et al, 2004) and yerosol inoculation (Cheville & Arp, 1978). Bacter- ial factors mediating translocation across the mucosal surfaces are ill-defined. In human exPEC, penetration of the blood brain barrier has boen shown to be mediated bby multiple factors that include ibe gene cluster (Kim, 2001). Recent work indicates that TheA may be playing 2 similar role in APEC 02 strains (Germon et al, 2005). In support of this, genetic surveys by PCR have established a link between the ibed gene and APEC (02 strains (Germon et al., 2005; Vidotto et al, 2007) However, the low frequency of ibed in APEC O78 strains (Germon et af., 2005) and its presence in someisolates from apparently healthy birds (Rodriguez-Sick et 4al., 2005a) implies an inconsistent requirement of this ‘gene For invasion, ‘Another potential invasin identified in APEC is the homologue of the 25 kDa Tia protein encoded by the tia locus in enterotoxigenic £. coli, The tia locus influences adherence and invasion of cultured human ileocaccal and colonic epithelial cells by enterotoxigenic £ cul! (Fleckenstein er al, 1996). In APEC, the ria gene is located on a S6 kb pathogenicity island termed PAL Igrecot: Which also carries the pap operon among other putative virulence factors (Kariyawasam et al, 20060), ‘This gene had been found to be significantly associated with strains from diseased birds (Kariyawasam e¢ al, 20066), Besides mediating adherence to chick explant tissues, ccurli have been reported to mediate invasion of eukar- yotie cells (Gophna er al, 2001) and invasion in the I~ ‘day-old chick infection model (La Ragione etal, 20000). However, the role in invasion per se is questionable since curl are widespread among non-pathogenic and labora tory-adapted strains, Iron acquisition systems. The ability of pathogenic bacteria to sequester iron from body fluids is considered pivotal for virulence, and in APEC this characteristic has been linked with lethality for I-day-old chicks (Dho & Lafont, 1984). Although a number of iron acquisition mechanisms exist in Gram-negative pathogens, the aerobactin system is by far the most well characterized ‘The system comprises genes for the synthesis of the hydroxamate siderophore aerobactin (ue) and for Ferrie acrobactin uplake (iu), and these are expressed. by Virulent strains of E colt (Warner et al, 1981; Lafont press in Ecol K-12 ‘nd transfer to fter Principle of sgnatur-tageed transposon mutagenesis important antigens in other bacterial pathogens; K. pneumoniae (Kurupati et a, 2006) and uropathogenic E. coli (Hagan & Mobley, 2007). Surtuce-lovalized antigens are likely (0 play an important role in patho- genesis and also stimulate protective immunity; hence the process can be focused on sheared or outer membrane ‘components from the eel surfaee, This strategy has led to the identification of 110 surface proteins of Campy Tobacter jemi, eight of which were further evaluated for protective efficacy in the mouse infection model (Pro- Khorova er af, 2006), In cases where convalescent sera eannot be obtained, surface-exposed proteins can be identified using a similar proteomie approach following labelling of bacterial cell surface with biotin, followed by detection of labelled proteins using streptavidin conju- gated to a fluorochrome or enzyme. Although the authors have selected to highlight STM and IVIAT, it should be emphasized there are other alternative genome-based approaches equally capable of Probe expression lbary + Screen sera from APEC:nfectd turkeys and chickens agaist expression Iiraries + deny immune reactive cones Figure 2 date firings and assas vaccine pont In vivo.indueed antigen technology:362. F, Driva and M. P, Stevens fining APEC activity in vivo, These include in vivo expression technology (reviewed in Rediers etal. 2005), transposon-site hybridization (Chan er al, 2005) and reporter gene fusions to dotect bacterial gene expression in vivo (Chalfic et al, 1994). The availability of the APEC O1 genome sequence (Johnson eal, 2007a) aso allows automatic in silico prediction of novel virulence loci, which would require validation for their roles in pathogenesis. The advent of high-throughput pyrose- {quencing methods promises the availability of many ‘more APEC genomes in years to come. ‘Concluding remarks Colibacillosis may be controlled by antimicrobial agents, >but the encounter of residues of stich agents in food is of major concern (Singer & Hofaere, 2006). Control by non-antibjoti interventions therefore presents an alter- native rational approach, Utilization of bacteriophages for the control of APEC has been explored, but this remained unpopular (Huff er al, 2003, 2005). Several ‘other alternatives for interventions to reduce carriage of pathogens in food-producing animals have been dis- ‘cussed elsewhere (Doyle & Erickson, 2006) and i is clear that vaccination provides the most rational option. In previous studies, vaccination with live attenuated mu- lants generated serotype-specific and steain-specific protection (Kwaga et al, 1994; Peighambari et al., 2002; Kariyawasam et al, 20046), hence the need t© develop cross-protective vaccines. The search for such vucines should remain at the forefront of future APEC research and, by combining bioinformatics, genomic and proteomic approaches, it can be expected that more ‘vaccine candidates will be identified. Promisingly, these approaches are already proving successful in the identi- fication of novel vaccine candidates in other pathogenic bacteria, Recent comparative genomic analysis led to the identification of candidates for subunit vaccines against exPEC (Durant et al, 2007). Following the successful application of reverse vacsinology to identify potential ‘vaccine candidates for Neisseria meningivids (Pizza et al, 2000), multiple genomic screening enabled the identification of a universal Group B Streptococcus ‘vaccine candidate (Maione er al., 2005). Furthermore, software for in silico identification of best vaccine candidates from the whole proteomes of bacterial pathogens has been developed (Vivona et al., 2006), Sequencing of the genomes of more APEC’ strains representing all predominant pathogenic serotypes will aid the reverse vaccinology approach. However, it should be remembered the predominant APEC serotypes O1, (02 and O78 potentially employ different mechanisms at different stages of infection (Mokady er al, 2005: Ron, 2006), which could pose a barrier to the design of eross- protective vaccines. AAs for other pathogens, a key challenge in the post- ‘genomic era will be to define the role af the encoded: APEC genes in pathogenesis and transmission, and to identify cellular constituents able 10 elicit protective immunity. For such a heterogeneous group of pathogens, it will be important to define the impact of variation in the repertoire, sequence and expression of encoded gene iI we are to derive broadly cross-protective intervention strategies in the future Acknowledgements “The present work was supported by The Biotechnology and Biological Seiences Research Couneil (Grant Ref No. BB/E001661/1), the British Poultry Council (Turkey RAD Sector) and Aviagen Lid, The authors thank Mick Gill for excellent technical assistance. References Achiman, M, Heazenoser, M., Kasesk, Bh, Osho, H., Cavan 1, Scns, RK. Vulnen- Re, V, Korhonen, TK. Sat, §. (akon, FA Orso, (986), Clonal analysis of Bieherohia call (OKI isolated from discus humans and animals. fection and Ino, 31, 268-26, Adis 'S. 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