Rabbit Enteropathogenic Escherichia Coli Research Articles

A directed intimin insertion mutant of a rabbit enteropathogenic Escherichia coli (REPEC) is attenuated, immunogenic and elicits serogroup specific protection

We previously showed (Agin et al., 2005) that a truncated beta-intimin mutant of an O15 A/E REPEC strain, which does not contain the C terminal tir binding region of intimin but expresses the preceding immunodominant portion of the molecule in outer membranes, is attenuated, induces anti-intimin and anti whole cell antibody, and protects against challenge with a virulent strain of the same serogroup. Since the ability of this and other intimin mutants lacking the tir binding region to provide broad protection against challenge with REPEC strains of other serogroups is incompletely studied, we generated a targeted insertion/deletion intimin mutant in an O103 strain, immunized rabbit's orogastrically, then challenged them with the parent strain and a virulent strain of a different serogroup. We used λ red recombinase to generate an eae mutation in the prototypic rabbit A/E Escherichia coli strain E22 (O103) by replacing the 81 C-terminal (860–939) tir-binding amino acids of intimin with an inserted kanamycin resistance gene. This mutant did not express intimin in its outer membranes. A range of increasing immunizing doses (104–107CFU) was used for the first immunization in 4 groups of 6 rabbits. All 4 groups received a 2nd immunization with 107CFU after 2weeks. At 4weeks, half of the rabbits in each group, and 6 control rabbits, were challenged with the parent O103 strain or with the O15 strain RDEC-H19A. All unimmunized rabbits exhibited characteristic weight loss with diarrhea and shedding of the challenge strain after challenge with E22 or RDEC-H19A. Rabbits challenged with the parent O103 E22, but not with the O15 RDEC-H19A, were protected against clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organism. At sacrifice, CFU of E22, but not RDECH19, were decreased in ileum, cecum and colon. Serum antibodies to E22 somatic antigens, but not intimin, were detected in rabbits immunized with E22 Δeae860–939 and correlated with protection. An intimin insertion mutation replacing the tir binding region of O103 REPEC strain E22 with an antibiotic resistance gene was attenuated, induced antibody to whole bacteria but not to intimin, and yielded protection against challenge with the WT strain from which it was prepared but not against a virulent strain of another serogroup. These results suggest that intimin expression in outer membranes may be necessary to confer cross serogroup protection by inducing anti-intimin immunity.

Virulence Inhibition by Zinc in Shiga-Toxigenic Escherichia coli

Previously, our laboratories reported that zinc inhibited expression of several important virulence factors in enteropathogenic Escherichia coli (EPEC) and reduced EPEC-induced intestinal damage in vivo. Since EPEC is genetically related to Shiga-toxigenic E. coli (STEC), we wondered whether the beneficial effects of zinc extended to STEC as well. Treatment options for STEC infection are very limited, since antibiotics tend to exacerbate disease via enhanced toxin production, so a safe intervention for this infection would be welcome. In this study, we report that in STEC strains zinc inhibits adherence to cultured cells as well as expression of EHEC secreted protein A (EspA). In addition, zinc inhibits the expression of Shiga toxin (Stx) at both the protein and the RNA level. Zinc inhibits basal and antibiotic-induced Stx production and inhibits both Stx1 and Stx2 by ≥90% at a concentration of 0.4 mM zinc. Rabbit EPEC strains were selected for acquisition of Stx-encoding bacteriophages, and these rabbit STEC strains (designated RDEC-H19A and E22-stx2) were used to test the effects of zinc in vivo in ligated rabbit intestinal loops. In vivo, zinc reduced fluid secretion into loops, inhibited mucosal adherence, reduced the amount of toxin in the loops, and reduced STEC-induced histological damage (villus blunting). Zinc has beneficial inhibitory effects against STEC strains that parallel those observed in EPEC. In addition, zinc strongly inhibits Stx expression; since Stx is responsible for the extraintestinal effects of STEC infection, such as hemolytic-uremic syndrome (HUS), zinc might be capable of preventing severe sequelae of STEC infection.

EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability

Many pathogenic bacteria subvert normal host cell processes by delivering effector proteins which mimic eukaryotic functions directly into target cells. EspF is a multifunctional protein injected into host cells by attaching and effacing pathogens, but its mechanism of action is not understood completely. In silico analyses of EspF revealed two key motifs: proline-rich domains and PDZ domain binding motifs. Such functional domains may allow EspF to act as an actin nucleation-promoting factor by mimicking host proteins. In agreement with these predictions, we found that EspF from rabbit enteropathogenic Escherichia coli (E22) participates in the regulation of actin polymerization by binding to a complex of proteins at the tight junctions (TJ). EspF bound to actin and profilin throughout the course of infection. However, after 2 h of infection, EspF also bound to the neural Wiskott-Aldrich syndrome protein and to the Arp2/3, zonula occludens-1 (ZO-1), and ZO-2 proteins. Moreover, EspF caused occludin, claudin, ZO-1, and ZO-2 redistribution and loss of transepithelial electrical resistance, suggesting that actin sequestration by EspF may cause local actin depolymerization leading to EspF-induced TJ disruption. Furthermore, EspF caused recruitment of these TJ proteins into the pedestals. An E22 strain lacking EspF did not cause TJ disruption and pedestals were smaller than those induced by the wild-type strain. Additionally, the pedestals were located mainly in the TJ. The overexpression of EspF caused bigger pedestals located along the length of the cells. Thus, actin sequestration by EspF allows the recruitment of junctional proteins into the pedestals, leading to the maturation of actin pedestals and the disruption of paracellular permeability.

Testing the efficacy of medium chain fatty acids against rabbit colibacillosis

Enteropathogenic Escherichia coli (EPEC) represents a major cause of lethal diarrhea in young mammals. Although the pathogenicity mechanisms of EPEC are now well understood, the intrinsic and environmental factors that control the expression of EPEC virulence remain largely unknown. In the rabbit, suckling reduces pups’ sensitivity to EPEC infection. Hence, we have hypothesized that uncharacterized factors present in doe milk may mediate this protection. Medium chain fatty acids (MCFA), known to possess antimicrobial properties, are highly abundant in doe milk. We demonstrate that caprylic acid exhibits a clear bacteriostatic effect in vitro against the rabbit EPEC strain E22 (O103:H2:K-), in a dose-dependent manner. In vivo, the dietary inclusion of triglycerides of MCFA did not however reduce the sensitivity of young rabbits challenged with this EPEC strain. The mortality and fecal excretion of EPEC were not reduced, and the bacterial adhesion to ileum was not inhibited. Amount of MCFA reaching the ileal level might have been too low and/or their association to other milk antimicrobials may have been required to observe a positive effect on disease evolution in a context of a highly virulent challenge.

The possible influence of LuxS in the in vivo virulence of rabbit enteropathogenic Escherichia coli

Attaching and effacing (A/E) organisms, such as rabbit enteropathogenic Escherichia coli (EPEC), human EPEC or enterohemorrhagic E. coli (EHEC) share attaching and effacing phenotype and LEE pathogenicity island responsible for A/E. The present study was undertaken to investigate the impact of the LuxS quorum sensing (QS) signaling system in vitro and in vivo pathogenicity of A/E organisms using rabbit EPEC (rEPEC) strain E22 (O103:H2). Analysis of the bioluminescence indicated abolished production of the QS signal AI-2 by luxS mutant (E22Δ luxS). Strain E22Δ lux also exhibited impaired expression of several normally secreted proteins and reduced adherence to cultured HeLa cells. Complementation of the intact luxS gene to E22Δ luxS restored secreted protein expression comparable to the WT type but not adherence to HeLa cells. In experimentally infected rabbits, the isogenic luxS mutant induced clinical illness and intimate adherence to the intestinal mucosa, albeit to a less extent, comparable to that seen with the parent virulent strain. It is worth noting that reduced fecal bacterial shedding, mucosal adherence and improved cumulative weight gain were seen for the mutant strain complemented with luxS when compared to the WT. It appears that the luxS gene is not essential for in vivo pathogenicity by rEPEC where exogenous QS signals are present in the gut. The impact of AI-2 provided by multicopy plasmid on bacterial virulence is discussed.

LEE-encoded regulator (Ler) mutants elicit serotype-specific protection, but not cross protection, against attaching and effacing E. coli strains

We previously showed that single dose orogastric immunization with an attenuated regulatory Lee-encoded regulator ( ler) mutant of the rabbit enteropathogenic Escherichia coli (REPEC) strain E22 (O103:H2) protected rabbits from fatal infection with the highly virulent parent strain. In the current study we assessed the degree of homologous (serotype-specific) and heterologous (cross-serotype) protection induced by immunization with REPEC ler mutant strains of differing serotypes, or with a prototype strain RDEC-1 (O15:H-) which expresses a full array of ler up-regulated proteins. We constructed an additional ler mutant using RDEC-1 thus, permitting immunization with a ler mutant of either serotype, O15 or O103, followed by challenge with a virulent REPEC strain of the same or different serotypes. Consistent with our previous data, the current study demonstrated that rabbits immunized with a RDEC-1 ler mutant were protected from challenge with virulent RDEC-H19A (RDEC-1 transduced with Shiga toxin-producing phage H19A) of the same serotype. Rabbits immunized with RDEC-1 or E22 derivative ler mutants demonstrated significant increase in serum antibody titers to the respective whole bacterial cells expressing O antigen but not to the LEE-encoded proteins. However, immunization with the ler mutants of either E22 or RDEC-1 failed to protect rabbits from infections with virulent organisms belonging to different serotypes. In contrast, rabbits immunized with the prototype RDEC-1 were cross protected against challenge with the heterologous E22 strain as shown by normal weight gain, and the absence of clinical signs of disease or characteristic attaching and effacing (A/E) lesions. Immunization with RDEC-1 induced significantly elevated serum IgG titers to LEE-encoded proteins. We thus, demonstrated homologous protection induced by the REPEC ler mutants and heterologous protection by RDEC-1. The observed correlation between elevated immune responses to the LEE-encoded proteins and the protection against challenge with heterologous virulent REPEC strain suggests that serotype-non-specific cross protection requires the expression of, and induction of antibody to, LEE-encoded virulence factors.

Protection of rabbits against enteropathogenic Escherichia coli(EPEC) using an intimin null mutant

BackgroundDiarrhea and mortality resulting from infections with enteropathogenic Escherichia coli (EPEC) are of major economic importance in the rabbit meat industry. There is a growing need for an effective vaccine to cope with these problems and to reduce the use of antibiotics. EPEC are characterized by an attaching and effacing virulence mechanism. This is partly mediated by the intimate binding between an adhesin, called intimin, and a translocated receptor (Tir) of prokaryote origin. We constructed an intimin deletion mutant of the rabbit EPEC (REPEC) wild-type strain 97/241.6 (bio-/serogroup 3-/O15) and examined its protective capacity.ResultsAfter verifying its complete loss of virulence, we used the attenuated strain in vaccination-challenge experiments in which complete protection against a homologous, but virulent, strain was observed. The attenuated strain was able to persist in the intestinal lumen, where it elicited an immune response against EPEC-related virulence proteins, as was shown using an EspB-specific ELISA. Despite the priming of an immune response and the generation of specific antibodies, the intimin mutant was not able to fully protect rabbits against challenges with REPEC strains of other bio-/serogroups.ConclusionThese data indicate that protection against REPEC infections is at least partly bio-/serogroup dependent and a multivalent vaccine may be needed for protection against the full range of REPEC types. Such a combination vaccine may be developed using intimin null mutants, as the latter were clearly shown to be safe and effective against homologous infections.

Genetically engineered enteropathogenic Escherichia coli strain elicits a specific immune response and protects against a virulent challenge

Enteropathogenic Escherichia coli (EPEC), a major cause of severe disease with diarrhea in infants, is also involved in weaned rabbit colibacillosis. EPEC O103 is frequent in rabbit-fattening units of Western Europe. It causes high mortality and growth retardation, leading to substantial economic losses. We report here the construction by allelic exchange of an EPEC O103 strain mutated in espB and tir, two essential virulence genes. Upon live oral administration to weaned rabbits, the E22ΔTir/EspB mutant strain efficiently colonized the intestinal tract without any adverse consequences. The rabbits were challenged with the highly pathogenic parental strain E22. The mutant provided complete protection to rabbits and total resistance to intestinal colonization by E22. In addition, E22ΔTir/EspB strain induced a specific humoral response against the bacterial adhesin AF/R2. These Abs prevent bacterial attachment to epithelial cells in vitro. These results open the way for the development of an efficient vaccine strategy against rabbit EPEC infections.

New flow cytometric method to quantify the inhibition of enteropathogenic Escherichia coli adhesion by anti-adhesin antibodies.

Pathogenesis of enteropathogenic Escherichia coli (EPEC) infections can be divided in three stages. The first one is the intestinal colonization mediated by bacterial adhesins. The second and third stages are characterized by an intimate attachment of bacteria to the enterocytes. Little information is available on the specific immune response against EPEC. Here, we describe and validate a new approach to quantify the function of anti-EPEC adhesin antibodies (Abs). We developed a new method to quantify the function of anti-adhesin Abs by flow cytometry. We used pEGFP-E22 (a rabbit EPEC E22 strain expressing the GFP protein) and HeLa cells. The adhesion of E22 bacteria to HeLa cells is mediated by AF/R2, the specific E22 adhesin. We performed short-time interaction (30 min) between pEGFP-E22 and HeLa cells. After extensive washes, 10,000 HeLa cells were acquired by flow cytometry and bacterial adhesion was quantified. Different sera were used to inhibit bacterial adhesion and recombinant MPB-Afr2G (Afr2G is the main AF/R2 subunit) was also tested in this system. We first verified that GFP expression by E22 did not modify bacterial adhesion. We then showed that this flow cytometry approach allowed easy quantification of bacterial adhesion and inhibition mediated by a specific anti-AF/R2 serum. Moreover, recombinant AF/R2 protein reversed the effect of the anti-AF/R2 serum. Finally, we validated our method using sera from E22 orally infected rabbits. We detected and quantified with this method functional specific anti-AF/R2 Abs in their sera. In addition, we correlated our results with an anti-AF/R2 enzyme-linked immunosorbent assay. We have developed a new method to detect and quantify specific anti-EPEC adhesin Abs by flow cytometry. This method is easy to use and highly reproducible. Its development could be extended to the search of specific anti-adhesin Abs in human EPEC infections.

Type III secretion-dependent cell cycle block caused in HeLa cells by enteropathogenic Escherichia coli O103.

Rabbit enteropathogenic Escherichia coli (EPEC) O103 induces in HeLa cells an irreversible cytopathic effect characterized by the recruitment of focal adhesions, formation of stress fibers, and inhibition of cell proliferation. We have characterized the modalities of the proliferation arrest and investigated its underlying mechanisms. We found that HeLa cells that were exposed to the rabbit EPEC O103 strain E22 progressively accumulated at 4C DNA content and did not enter mitosis. A significant proportion of the cells were able to reinitiate DNA synthesis without division, leading to 8C DNA content. This cell cycle inhibition by E22 was abrogated in mutants lacking EspA, -B, and -D and was restored by transcomplementation. In contrast, intimin and Tir mutants retained the antiproliferative effect. The cell cycle arrest was not a direct consequence of the formation of stress fibers, since their disruption by toxins during exposure to E22 did not reverse the cell cycle inhibition. Likewise, the cell cycle arrest was not dependent on the early tyrosine dephosphorylation events triggered by E22 in the cells. Two key partner effectors controlling entry into mitosis were also investigated: cyclin B1 and the associated cyclin-dependent kinase 1 (Cdk1). Whereas cyclin B1 was not detectably affected in E22-exposed cells, Cdk1 was maintained in a tyrosine-phosphorylated inactive state and lost its affinity for p13(suc1)-agarose beads. This shows that Cdk1 is implicated in the G2/M arrest caused by EPEC strain E22.

Decreased apoptosis in the ileum and ileal Peyer's patches: a feature after infection with rabbit enteropathogenic Escherichia coli O103.

Significant changes occur in intestinal epithelial cells after infection with enteropathogenic Escherichia coli (EPEC). However, it is unclear whether this pathogen alters rates of apoptosis. By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103). Various REPEC O103 strains were first tested in vitro for characteristic virulence features. Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently. After experimental infection, intestinal samples were examined by light and electron microscopy. Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining). The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; P = 0.01), in the tips of ileal absorptive villi (determined by H&E staining; P = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; P = 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum.

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

The long-term cytoskeletal rearrangement induced by rabbit enteropathogenic Escherichia coli is Esp dependent but intimin independent.

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.

Two Enteropathogenic Escherichia coli Type III Secreted Proteins, EspA and EspB, Are Virulence Factors

Enteropathogenic Escherichia coli (EPEC) belongs to a family of related bacterial pathogens, including enterohemorrhagic Escherichia coli (EHEC) O157:H7 and other human and animal diarrheagenic pathogens that form attaching and effacing (A/E) lesions on host epithelial surfaces. Bacterial secreted Esp proteins and a type III secretion system are conserved among these pathogens and trigger host cell signal transduction pathways and cytoskeletal rearrangements, and mediate intimate bacterial adherence to epithelial cell surfaces in vitro. However, their role in pathogenesis is still unclear. To investigate the role of Esp proteins in disease, mutations in espA and espB were constructed in rabbit EPEC serotype O103 and infection characteristics were compared to that of the wild-type strain using histology, scanning and transmission electron microscopy, and confocal laser scanning microscopy in a weaned rabbit infection model. The virulence of EspA and EspB mutant strains was severely attenuated. Additionally, neither mutant strain formed A/E lesions, nor did either one cause cytoskeletal actin rearrangements beneath the attached bacteria in the rabbit intestine. Collectively, this study shows for the first time that the type III secreted proteins EspA and EspB are needed to form A/E lesions in vivo and are indeed virulence factors. It also confirms the role of A/E lesions in disease processes.

Proliferative enterocolitis associated with dual infection with enteropathogenic Escherichia coli and Lawsonia intracellularis in rabbits.

Both enteropathogenic Escherichia coli (EPEC) and an obligate intracellular bacterium, previously referred to as an intracellular Campylobacter-like organism and now designated Lawsonia intracellularis, have been reported as causes of enterocolitis in rabbits. An outbreak of enterocolitis in a group of rabbits, characterized by an unusually high rate of mortality, was found to be associated with dual infection with EPEC and L. intracellularis. The EPEC strain was found to have eaeA gene homology but was negative for afrA homology. The absence of the afrA gene, which encodes the structural subunit for the AF/R1 pilus, indicates that this rabbit EPEC strain is distinct from the prototypic RDEC-1 strain. This finding suggests that rabbit EPEC strains widely reported in Western Europe, which lack AF/R1 pili, are also present in rabbits in the United States. Dual infection with these two pathogens in rabbits has not been previously reported and may have contributed to the unusually high mortality observed in this outbreak.

Identification and characterization of a K88- and CS31A-like operon of a rabbit enteropathogenic Escherichia coli strain which encodes fimbriae involved in the colonization of rabbit intestine.

Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.

Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits.

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.

Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal expression is sensitive to host body temperature.

Enteropathogenic Escherichia coli (EPEC) and rabbit EPEC (RDEC-1) cause unique histopathological features on intestinal mucosa, including attaching/effacing (A/E) lesions. Due to the human specificity of EPEC, RDEC-1 has been used as an animal model to study EPEC pathogenesis. At least two of the previously identified EPEC-secreted proteins, EspA and EspB, are required for triggering host epithelial signal transduction pathways, intimate adherence, and A/E lesions. However, the functions of these secreted proteins and their roles in pathogenesis have not been characterized. To investigate the function of EspA and EspB in RDEC-1, the espA and espB genes were cloned and their sequences were compared to that of EPEC O127. The EspA proteins showed high similarity (88.5% identity), while EspB was heterogeneous in internal regions (69.8% identity). However, RDEC-1 EspB was identical to that of enterohemorrhagic E. coli serotype O26. Mutations in RDEC-1 espA and espB revealed that the corresponding RDEC-1 gene products are essential for triggering of host signal transduction pathways and invasion into HeLa cells. Complementation with plasmids containing EPEC espA or/and espB genes into RDEC-1 mutant strains demonstrated that they were functionally interchangeable, although the EPEC proteins mediated higher levels of invasion. Furthermore, maximal expression of RDEC-1 and EPEC-secreted proteins occurred at their respective host body temperatures, which may contribute to the lack of EPEC infectivity in rabbits.

Intestinal Colonisation with Different Rabbit EnteropathogenicEscherichia coliBiotypes and Cross-Protection Induced by Different Strains

Experimental infections with four different rabbit enteropathogenic Escherichia coli (EPEC) biotypes were carried out in EPEC free rabbits, and intestinal colonisation was assessed by measuring semi-quantitatively the rectal colonisation. When the animals were infected for the first time shortly after weaning, the excretion followed an approximately identical course with all four EPEC: at 3-9 d post-infection all rabbits excreted the infecting EPEC strain in high quantities, and continued to do so for 1-2 wks. Rabbits that had survived such a colonisation with one EPEC, were protected against colonisation with strains belonging to other EPEC groups. Keywords: E. coli ; Rabbit EPEC; Intestinal colonisation; Cross-protection.