MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium.

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli chi7122 conferred mannose-resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain chi7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low-copy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature- and RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.

Cytokine mRNA expression in pigs infected with Schistosoma japonicum.

The pig is a natural host of Schistosoma japonicum and a useful animal model of human disease. In the present study mRNA levels of Th1 (IFN-gamma) and Th2 (IL-10 and IL-4) cytokines were assessed by RT-PCR within tissues from infected pigs. Twelve Danish crossbred pigs were infected by intramuscular injection or orally with 1000 cercariae. Six other pigs served as non-infected controls. Liver and intestinal tissues were collected 10 weeks post-infection, and analysed for their relative levels of cytokine mRNA. Infected pigs developed a Th2 response as characterized by the increased level of mRNA encoding for IL-4 and IL-10 in their large intestine (caecum and colon). In contrast, levels of IFN-gamma did not differ between control and infected animals although variation between animals was observed. When comparing the immune response of orally and intramuscularly infected animals, we found that orally infected pigs produced higher IL-4 and IL-10 levels in their caecum and colon respectively. This stronger Th2 response correlated with a previously reported delay in maturation of infection following oral infection. The cytokine expression levels in tissue samples taken from lesion sites and in nearby areas, without obvious lesions, were then compared. Subsequent to an oral infection, the Th2 type cytokine production was higher in the lesion sites of the liver. In conclusion, this study is the first demonstration of IL-4 and IL-10 cytokine response in pig tissues during S. japonicum infection.

Relationship between the Tsh autotransporter and pathogenicity of avian Escherichia coli and localization and analysis of the Tsh genetic region.

The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain chi7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with chi7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain chi7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain chi7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.

Cloning, chromosomal location, and tissue expression of the gene for pig interleukin-18.

Interleukin 18 (IL-18), a recently described cytokine, plays an important role in the cell-mediated immune response, in particular through its ability to induce the production of interferon (IFN)-gamma. We cloned pig IL-18 cDNA from the intestinal epithelial cell line IPI-2I using a reverse transcriptase-polymerase chain reaction method with primers derived from the human IL-18 sequence. The amino acid sequence deduced from pig IL-18 cDNA encodes a 192 amino-acid polypeptide that exhibits 92, 90, 81, and 71% similarity to IL-18 from horse, dog, human, and rodents (mouse and rat), respectively. Structural comparison of the IL-18 protein with IL-1alpha and IL-1beta showed that IL-18 shares several characteristics with the IL-1 cytokine family: the IL-1 signature-like sequence, a potential caspase-1 (ICE) cleavage site, and the presence of 12 predicted beta strands. Fluorescence in situ hybridization was used to localize the IL-18 gene on the short arm (p13) of pig chromosome 9. Analysis of IL-18 expression in different organs of piglets demonstrated that IL-18 mRNA is weakly expressed in the kidney and the lung. By contrast, we observed highly constitutive expression of IL-18 mRNA in the spleen, mesenteric lymph nodes, and the intestine, particularly in the small intestine, indicating a potential role for IL-18 as a first line of host defense in the intestinal mucosa.

Lack of a role of cytotoxic necrotizing factor 1 toxin from Escherichia coli in bacterial pathogenicity and host cytokine response in infected germfree piglets.

Some Escherichia coli strains isolated from intestinal or extraintestinal infections in pigs produce cytotoxic necrotizing factor 1 (CNF1). In order to analyze the role of CNF1 in the pathogenesis of porcine colibacillosis, newborn colostrum-deprived germfree piglets were orally inoculated with a wild-type CNF1-producing strain (M623) or with an isogenic cnf1 mutant (M623DeltaCNF1). The two isogenic strains induced a high mortality with similar lung and serosal inflammatory lesions, indicating that both strains were pathogenic in these piglets. Bacterial counts in various organs of inoculated piglets revealed an intestinal predisposition of M623 and M623DeltaCNF1 strains for the cecum and colon. Extraintestinal organs (lungs, liver, spleen, and kidney) were also colonized by both strains. Similar colonization of intestinal and extraintestinal tissues in animals inoculated with either strain was observed, except in the ileum, where M623 showed a higher colonization than M623DeltaCNF1. Intestinal (ileum and colon), extraintestinal (lung and kidney), and immune (mesenteric lymph nodes and spleen) tissues were sampled at 1 day postinoculation and analyzed for cytokine expression by a reverse transcriptase PCR technique. Inoculation with E. coli M623 induced an enhanced expression of inflammatory cytokines (interleukin-1alpha [IL-1alpha], tumor necrosis factor alpha, and IL-12p40) in the intestinal organs compared to uninoculated piglets or piglets inoculated with nonpathogenic intestinal E. coli 862B, which is also able to colonize the intestinal tract. There was little difference in cytokine transcript levels in the intestinal and extraintestinal organs in piglets inoculated with E. coli strains M623 or M623DeltaCNF1, except in the ileum, where IL-1alpha and IL-8 mRNA levels correlated with bacterial colonization. Expression of regulatory cytokines (gamma interferon and IL-4) was weak in immune tissues from piglets inoculated with M623 or M623DeltaCNF1. Taken together, our data indicate that the CNF1-producing strain, M623, is pathogenic and induces inflammatory cytokine expression in germfree, colostrum-deprived piglets. Nevertheless, in this model, the CNF1 toxin does not appear to be a major factor for pathogenicity or cytokine response, as demonstrated by the use of an isogenic cnf1 mutant.

Tumor necrosis factor is required for the priming of peritoneal macrophages by trehalose dimycolate.

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. We have developed an original model of macrophage activation where TDM is injected in vivo to prime peritoneal macrophages. These primed macrophages do not express inducible NO synthase (NOS II), however, they can be fully activated, i.e. induced to express NOS II and to develop a NOS II-dependent antiproliferative activity, following in vitro exposure to low concentrations of LPS. In a previous paper, we have shown that TDM-priming of mouse peritoneal macrophages is mediated by the sequential production of IL-12 and IFN-gamma. In the present paper, we investigated the role of TNF in the priming of macrophages by TDM. By semi-quantitative RT-PCR, we have shown that TDM injection induced transcription of TNF-alpha in peritoneal cells. TNF-mRNA levels peaked 5 hours after TDM injection and remained elevated for at least 32 hours. TNF expression was absolutely necessary for macrophage priming, as injection of an anti-TNF monoclonal antibody, 4 h before and 20 hours after TDM injection, prevented LPS-dependent activation of macrophages in vitro. This result was confirmed by the inability of TDM to prime macrophages from LT-alpha/TNF-alpha knockout (LT/TNFKO) mice. In addition, analysis of LT/TNFKO mice treated with TDM revealed that induction of the IL-12 transcript in their peritoneal cells and expression of a functional NADPH oxidase in macrophages are TNF-independent events.

Pathogenic diversity of Escherichia coli and the emergence of 'exotic' islands in the gene stream.

Escherichia coli is a highly adaptive bacterial species that is both a member of the commensal intestinal flora and a versatile pathogen associated with numerous types of intestinal and systemic infections in humans and other animals. The spectrum of diseases caused by E. coli is due to the acquisition of specific virulence genes harbored on plasmids, bacteriophages, or within distinct DNA segments termed pathogenicity islands (PAIs) that are absent from the genomes of commensal E. coli strains. PAIs are likely to have been transferred horizontally and may have integrated into the E. coli chromosome through bacteriophage or plasmid integration or transposition. The contribution of intergenic inheritance to the adaptation and evolution of E. coli, types of PAIs associated with different groups of pathogenic E. coli and approaches to identify unique sequence islands (USIs), some of which might confer pathogenicity, in E. coli and other bacteria are presented.

Expression of P, S, and F1C adhesins by cytotoxic necrotizing factor 1-producing Escherichia coli from septicemic and diarrheic pigs.

Nineteen papC-positive cytotoxic necrotizing factor 1 (CNF1)-producing Escherichia coli isolates from pigs with septicemia or diarrhea were tested for the presence of pap-, sfa-, and afa-related sequences encoding P/Prs, S/F1C, and Dr/AFA adhesins respectively. Production of adhesins by isolates was tested by mannose-resistant hemagglutination (MRHA), sialidase treatment of erythrocytes and particle agglutination tests. Production of P, S, and F1C fimbriae by isolates was also examined by immunofluorescence. All isolates were pap+ by PCR. Eighteen isolates (95%) were MRHA for ovine and human A erythrocytes and exhibited GalNac-GalNac receptor specificity associated with class III P(Prs) adhesins. Fifteen (79%) of the 19 isolates reacted with antisera specific for one or more different P fimbrial serotypes on immunofluorescence. Three of these isolates also demonstrated Gal-Gal receptor specificity associated with class I or II P fimbrial adhesins. Fifteen (79%) of the isolates were sfa+ by PCR. Seven of these isolates exhibited sialidase-sensitive MRHA of bovine and human O erythrocytes and reacted with serum specific for S fimbriae on immunofluorescence. Seven of the 8 sfa+ isolates which were MRHA-negative for bovine erythrocytes reacted with serum specific for F1C fimbriae on immunofluorescence. All isolates produced type 1 fimbriae as determined by mannose-sensitive agglutination of yeast cells. None of the isolates were afa+ by PCR or colony hybridization. Results suggest that most pap+ porcine CNF1-producing E. coli isolates express P fimbriae bearing class III (Prs) type adhesins. In addition, most of these isolates also produce S or F1C fimbriae.

Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages.

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. TDM primes murine macrophages (Mphi) to produce nitric oxide (NO) and to develop antitumoral activity upon activation with low doses of lipopolysaccharide (LPS). In this study, we investigated the ability of TDM to induce interleukin 12 (IL-12) and the role of this cytokine in TDM-induced activation of murine Mphi. RNA isolated from peritoneal exudate cells (PEC) collected at different times after TDM injection was used to determine IL-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA levels by semiquantitative reverse transcriptase-PCR. Constitutive expression of IL-12p35 was observed in PEC from untreated as well as from TDM-injected mice. In contrast, expression of the IL-12p40 subunit was almost undetectable in control PEC but was dramatically upregulated in PEC from TDM-injected mice. IL-12p40 expression peaked at 8 h and subsided to baseline levels at 39 h postinjection. TDM was also able to induce IFN-gamma expression; however, kinetics of induction of IFN-gamma was different from that of IL-12p40. Maximal levels of IFN-gamma mRNA were reached by 24 h and did not return to baseline by 4 days. In addition, pretreatment of mice with neutralizing monoclonal antibodies directed against IL-12 (C15.6.7 and C15.1.2) blocked IFN-gamma mRNA induction in PEC from TDM-treated mice. We further determined if the induction of IL-12 and/or IFN-gamma contributes to the in vivo priming effect of TDM on peritoneal Mphi. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-gamma (XMG.1.6) monoclonal antibodies. TDM-primed Mphi were then activated in vitro with LPS and tested for their ability to produce NO and to develop cytostatic activity toward cocultivated L1210 tumor cells. Priming of Mphi by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-gamma as demonstrated by an absence of tumoricidal activity and NO production by TDM-elicited Mphi in the presence of LPS. Taken together our results show that TDM, a defined molecule from M. tuberculosis, induces in vivo production of IL-12. Moreover, synthesis of IL-12 mediates TDM priming of mouse peritoneal Mphi through IFN-gamma induction.

Mutations in the f165(1)A and f165(1)E fimbrial genes and regulation of their expression in an Escherichia coli strain causing septicemia in pigs.

Transposon (TnphoA) mutagenesis was used to study the expression of F165(1) fimbriae, related to Prs fimbriae, in the pathogenic Escherichia coli strain 5131 (O115:K "V165":F165). This strain causes septicemia in swine and also expresses F165(2) fimbriae, related to F1C. Adhesin-defective mutants from the wild-type pathogenic strain were produced and TnphoA insertions were localized either in the f165(1)A gene, which encodes the major fimbrial subunit or in the f165(1)E, gene, which encodes a minor fimbrial subunit. TnphoA gene fusions were used to measure expression of F165(1) fimbrial genes. Similar pattern of regulation of expression was observed in both f165(1)A and f165(1)E genes. Optimal expression of F165(1) fimbriae was obtained on solid minimal medium. Production of F165(1) fimbriae was negatively regulated by addition of glucose, leucine or alanine to the media, by growth at 18 degrees C, and by pH above or below 7.0.

Dynamics of Escherichia coil infection in experimentally inoculated chickens.

In order to study the dynamics of avian colibacillosis, commercial broiler chickens were inoculated with a pathogenic Escherichia coli strain (01:K1:H7) into the left caudal thoracic air sac. Chickens were euthanatized at different times from 3 to 48 hr postinoculation and examined for bacterial counts and macroscopic and microscopic lesions. The E. coli strain colonized the air sacs, lungs, and trachea and was recovered from blood and all tested extrarespiratory organs of inoculated birds. A gradual increase in bacterial counts in the trachea, lungs, air sacs, and liver was observed from 3 to 12 hr. Clinical signs and macroscopic lesions of colibacillosis were observed in all inoculated birds. Moderate to severe lesions of airsacculitis, pericarditis, perihepatitis, and splenic hypertrophy were observed. Microscopically, inflammatory cell infiltration, serious to fibrinous exudate, and cellular debris on serosal surfaces were present in the liver, spleen, and air sacs. In air sacs, heterophils were present in low numbers perivascularly 3 hr after inoculation and became more numerous by 24 hr postinoculation. Ultrastructurally, epithelial cells in the air sacs and in air capillary regions of the lung were swollen and vacuolated beginning at 3 hr postinoculation. Bacteria were adherent to and present within the epithelial cells at 3 hr postinoculation and were also seen in phagocytic cells and, rarely, in the connective tissue of these organs at 24 hr postinoculation. These results indicate that both air sacs and lungs can be the portal of entry for E. coli into the systemic circulation, probably via damaged epithelium.

A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression.

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provide a highly sensitive, rapid, and simple means of simultaneously measuring the expression of porcine cytokines in immune cell populations. Oligonucleotide primers were designed to amplify porcine cytokine cDNA from genes encoding IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, TNF-beta and the housekeeping genes beta-actin and cyclophilin by PCR. Primers were chosen from different exons to detect for possible genomic DNA contamination of samples. To validate RT-PCR, unstimulated and concanavalin A (ConA) stimulated porcine peripheral blood mononuclear cells (PBMCs) were cultured from 2 h to 72 h, RNA was extracted and reverse transcribed, and cDNA was amplified using the different primer sets. Band intensities of PCR products were quantified by densitometric scanning and values were normalized against cyclophilin. For each of the cytokines, the kinetics of gene expression were similar among PBMCs isolated from different animals and could be grouped into two main patterns. Lymphocyte derived cytokines (IL-2, IL-4, IFN-gamma, and TNF-beta) exhibited low level expression in unstimulated cells and increased expression in ConA-stimulated PBMCs. IFN-gamma and IL-2 mRNA levels peaked at 24 h and returned to baseline by 72 h, whereas IL-4 and TNF-beta mRNA levels did not return to baseline by 72 h. In contrast, substantial mRNA levels for inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-12, and TNF-alpha) and IL-10 were detected from both unstimulated and ConA-stimulated PBMCs. Results indicate that RT-PCR is a sensitive and convenient method to monitor cytokine mRNA expression in porcine samples.

P-fimbriae-producing septicaemic Escherichia coli from poultry possess fel-related gene clusters whereas pap-hybridizing P-fimbriae-negative strains have partial or divergent P fimbrial gene clusters.

The organization of P fimbrial gene clusters of 13 papC-hybridizing Escherichia coli strains isolated from poultry with colisepticaemia, five P-fimbriae-expressing (P-positive) and eight P-fimbriae-non-expressing (P-negative), were examined by PCR and by Southern blot hybridization using primers or gene probes specific to the I, B, A, C or G genes. The absence of P fimbrial expression was associated with lack of PCR amplification of one or more of these genes, most commonly the I gene. Restriction endonuclease EcoRI, BamHI or PstI digests of genomic DNA from all strains hybridized with each of the gene probes and demonstrated polymorphisms between P-positive and P-negative strains. PstI digests of DNA from 12 of the 13 strains, when hybridized with the A gene probe, demonstrated a 0.1 kb fragment specific to the felA gene which encodes the major structural protein of F11 fimbriae. Hence, only the P-positive strains contained complete copies of fel-related gene clusters. In contrast, most of the pap-hybridizing P-negative strains contained partial or divergent P fimbrial gene clusters, which explains the lack of P fimbrial expression by these strains.

Expression of P and type 1 (F1) fimbriae in pathogenic Escherichia coli from poultry.

To investigate the expression of P and type 1 (F1) fimbriae in pathogenic avian Escherichia coli, fourteen pap+/fim+ E. coli isolates pathogenic for poultry were grown on four complex or minimal media, and examined for the presence of mannose resistant (MR) and mannose sensitive (MS) hemagglutination (HA), and for P or for type 1 (F1) fimbriae using immunofluorescence, immunodot, and immunoblot. In addition, isolates grown under different culture conditions were examined for adherence to frozen sections of chicken trachea. Twelve of the 14 isolates were divided into three groups based on adhesin expression in the different media. Isolates of all three groups exhibited strong MSHA reactions when cultures were grown serially in static broth, and expressed a subunit protein with an apparent molecular mass of 17 to 18.5 kDa, serologically related to the FIA major fimbrial subunit. There was a good correlation between MSHA and adherence to chicken tracheal sections. Isolates of group I only demonstrated MSHA and expression of F1A fimbriae after growth in static broth. Isolates of group II demonstrated MSHA and expression of F1A fimbriae after growth in all tested media whereas isolates of group III demonstrated expression of F1A fimbriae only after growth in static broth and minimal agar. Only the five group I isolates expressed MRHA associated with P fimbrial adhesins and expressed fimbriae with a major subunit protein of 18 kDa serologically related to the F11 major fimbrial subunit. None of these five isolates grown on complex solid media, where P but not type 1 fimbriae were expressed, adhered to tracheal sections. Results suggest that i) P fimbriae are not readily expressed in vitro by most pap+/fim+ avian E. coli isolates; ii) environmental control of phase variation of type 1 fimbriae differs among pathogenic avian E. coli; and iii) receptors for type 1, but not for P fimbriae, are present in chicken tracheal mucosa.

Bacterial colonization and in vivo expression of F1 (type 1) fimbrial antigens in chickens experimentally infected with pathogenic Escherichia coli.

Escherichia coli strains that cause septicemia of poultry often possess F1 (type 1) fimbriae (encoded by pil [fim] homologous gene clusters) and/or P fimbriae (encoded by pap homologous gene clusters). These fimbriae are thought to be involved in infection and colonization. To study the dynamics of infection due to E. coli with different virulence determinant profiles and to examine the expression of these fimbriae in vivo, three pathogenic E. coli isolates--O1 (pil+/pap+), O2 (pil+/pap), and O78 (pil+/pap+)--were administered intratracheally to 1.5-week-old chickens. Chickens were euthanatized from 3 to 144 hr after infection. The three isolates caused lesions in 30 to 55% of birds. Colonization rates of the trachea, lungs, internal organs, and pericardial fluid were similar for all three isolates, whereas significant differences among isolates were observed in colonization of the air sacs and blood. Bacteria appeared rapidly in the blood, liver, and spleen, whereas presence in the pericardial fluid generally occurred only after 24 hr postinoculation. The dynamics of colonization of the air sacs varied among isolates. Immunofluorescence of frozen tissue sections demonstrated F1 fimbriae (pil expressed) but not P fimbriae on all three isolates colonizing the trachea and on the O1 and O78 isolates colonizing the air sacs. Results suggest that F1 fimbriae are involved in the early stages of development of colisepticemia by promoting association of pathogenic E. coli with the trachea and air sacs of chickens.

pap-and pil-related DNA sequences and other virulence determinants associated with Escherichia coli isolated from septicemic chickens and turkeys.

Escherichia coli isolates from septicemic or healthy chickens and turkeys from Quebec were serotyped, examined genotypically by using DNA probes specific for the pil and pap fimbrial systems and the aerobactin siderophore system, and examined phenotypically for lethality in day-old chicks, hemagglutination, serum resistance, and aerobactin production. Serogroups O78 and O1 were most common in septicemic chickens and turkeys. pap+ isolates from chickens were associated with septicemia, and pap+ isolates from turkeys were associated with lethality in day-old chicks. Four of nine pap+ isolates from septicemic turkeys expressed P adhesin, whereas all pap+ isolates from septicemic chickens were negative for P adhesin. The pil+ genotype was associated with septicemia in chickens and with serum resistance in isolates from turkeys. Mannose-sensitive hemagglutination of guinea pig erythrocytes was associated with septicemia in chickens and turkeys, although this phenotype was not associated with pil+ isolates from turkeys. Serum resistance was associated with isolates from septicemic turkeys and with lethality in isolates from chickens. The aerobactin system was associated with isolates from septicemic chickens and turkeys. Overall, results indicated that (i) genotypic examination may reveal virulence-associated traits which differ from the typically expected phenotype and/or are not readily expressed in vitro, and (ii) certain phenotypic and genotypic traits associated with E. coli causing extraintestinal disease in humans and animals are also associated with E. coli causing avian septicemia.