Examination of Escherichia coli from poultry for selected adhesin genes important in disease caused by mammalian pathogenic E. coli.

A collection of 1601 extraintestinal and intestinal Escherichia coli isolated from chickens, turkeys and ducks, in Belgium, France and Spain, was hybridised with gene probes specific for fimbrial and afimbrial adhesins (F17, F18, S , Bfp, Afa, Cs31A, Intimin , Aida-1) of intestinal, urinary and invasive E. coli of mammals and with a probe specific for the P (Pap/Prs) fimbrial adhesin of urinary and invasive E. coli of mammals and birds. Three hundred and eighty-three strains (23.9%) were P-positive, 76 strains (4.8%) were Afa-positive, 75 strains (4.7%) were F17-positive, 67 strains (4.2%) were S-positive, 23 (1.4%) were Intimin-positive, and all were F18-, Cs31A-, Aida1- and Bfp-negative. The 75 F17-positive strains harboured different major subunit A-encoding gene variants, but the f17Ac variant was the most frequent (52 strains, 69.3%) and seven strains (9.3%) were not typeable. The f17G gene variant coding for the GII adhesin was the most frequent (56 strains, 75.0%), whereas the f17GI gene variant was present in four strains (5%) and 15 strains (20.0%) were not typeable. All Afa-positive strains harboured the afa-8 variant. The 23 Intimin-positive E. coli tested positive for the beta-variant (16 strains; 69.6%) or for the gamma-variant (seven strains; 30.4%) of the eae gene. Chicken and turkey E. coli were more frequently probe-positive (43.6 and 43.1%, respectively) than duck E. coli (31.5%) and extraintestinal E. coli were also more frequently probe-positive (48.4%) than intestinal strains (18.5%). Different combinations of probe positive hybridisation results were observed in 72 of the 540 probe-positive E. coli (13.3%). The most frequent combinations were between AfaE-8 and F17 probes (47 strains; 8.7%) and between P and S probes (13 strains; 2.4%). Although f17- and afa-8-related DNA sequences can be plasmid-located in mammalian E. coli, they were not in avian E. coli. Besides the P fimbrial adhesins, F17 and S fimbrial and Afa-VIII and Intimin afimbrial adhesins may thus represent colonisation factors of avian pathogenic E. coli.

Characterization of high-level fluoroquinolone resistance in Escherichia coli O78:K80 isolated from turkeys.

Fluoroquinolone resistance was characterized in Escherichia coli O78:K80 isolated from diseased turkeys. The level of resistance to fluoroquinolones of the isolates appeared closely correlated with substitutions in GyrA and ParC, but not with the production of the AcrAB efflux pump. Among isolates highly resistant to ciprofloxacin (MIC 8 mg/L) and harbouring identical substitutions (two in GyrA and one in ParC), two close but distinguishable ribotypes were identified. This indicated that at least two independent selection events may have occurred.

Increased tracheal colonization in chickens without impairing pathogenic properties of avian pathogenic Escherichia coli MT78 with a fimH deletion.

Several studies suggest that the expression of F1 fimbriae could be involved in the virulence of Escherichia coli for chickens. F1 fimbriae display multivalent properties such as adhesion to epithelia or interaction with the immune system that imply specific interactions between the adhesin FimH and different cell receptors. We constructed a delta fimH mutant of the avian pathogenic E. coli MT78 and evaluated its in vivo colonization and pathogenicity, as compared to that of the parent strain. The generated mutant PA68 was unable to adhere in vitro to chicken epithelial pharyngeal or tracheal cells; mutant bacteria were mostly afimbriated although a minority of them displayed altered piliation phenotypes. Two inoculation routes were used to compare the ability of MT78 and PA68 to colonize the respiratory tract and to induce colibacillosis in chickens. In the first model, 2-wk-old axenic chickens were inoculated intratracheally with one or both E. coli strains, after primary infection with infectious bronchitis virus. In the second model, 3-wk-old specific-pathogen-free chickens were inoculated via the caudal thoracic air sac. After intratracheal inoculation, the delta fimH mutant was found to be a better colonizer than MT78 in the trachea of inoculated chickens. Furthermore, when both strains were inoculated simultaneously, the delta fimH mutant constituted 98% of the bacterial population in the trachea at day 7 postinoculation. Irrespective to the inoculation route, MT78 and PA68 showed similar abilities to induce macroscopic lesions in chickens, to provoke bacteremia, and to colonize the internal organs. However, 4 days after intra-air sac inoculation, bacterial counts of the mutant were lower in the spleen and liver than those of MT78. Our results show that FimH is not required for colonization of the trachea of axenic chickens by E. coli and that it is not a major determinant of bacterial pathogenicity. On the contrary, the lack of expression of FimH seems to favor the in vivo colonization of the trachea of chickens by E. coli.

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.

Avian pathogenic Escherichia coli (APEC).

Avian pathogenic Escherichia coli (APEC) cause aerosacculitis, polyserositis, septicemia and other mainly extraintestinal diseases in chickens, turkeys and other avian species. APEC are found in the intestinal microflora of healthy birds and most of the diseases associated with them are secondary to environmental and host predisposing factors. APEC isolates commonly belong to certain serogroups, O1, O2 and O78, and to a restricted number of clones. Several experimental models have been developed, permitting a more reliable evaluation of the pathogenicity of E. coli for chickens and turkeys. Hence, virulence factors identified on APEC are adhesins such as the F1 and P fimbriae, and curli, the aerobactin iron sequestering system, K1 capsule, temperature-sensitive hemagglutinin (Tsh), resistance to the bactericidal effects of serum and cytotoxic effects. Experimental infection studies have shown that the air-exchange regions of the lung and the airsacs are important sites of entry of E. coli into the bloodstream of birds during the initial stages of infection and that resistance to phagocytosis may be an important mechanism in the development of the disease. They have also demonstrated that F1 fimbriae are expressed in the respiratory tract, whereas P fimbriae are expressed in the internal organs of infected chickens. The role of these fimbrial adhesins in the development of disease is not yet, however, fully understood. The more recent use of genetic approaches for the identification of new virulence factors will greatly enhance our knowledge of APEC pathogenic mechanisms. Diagnosis of APEC infections is based on the clinical picture, lesions and isolation of E. coli. This may be strengthened by serotyping and identification of virulence factors using immunological or molecular methods such as DNA probes and PCR. Approaches for the prevention and control of APEC infections include the control of environmental contamination and environmental parameters such as humidity and ventilation. Antibiotherapy is widely used, although APEC are frequently resistant to a wide range of antibiotics. Vaccines containing killed or attenuated virulent bacteria protect against infection with the homologous strain but are less efficient against heterologous strains. Hence, vaccination for colibacillosis is not widely practised because of the large variety of serogroups involved in field outbreaks.

Colonization ability and pathogenic properties of a fim- mutant of an avian strain of Escherichia coli.

Several studies suggest that the expression of type 1 fimbriae is involved in the virulence of Escherichia coli in chickens, by promoting adhesion of bacteria to the respiratory tract, which is most probably the first step to occur in the infection, and by interacting with the immune response. In order to determine to what extent type 1 fimbriae were involved in the pathogenic process, the fim cluster of an avian pathogenic strain of E. coli, MT78 (O2:K1:H+), was modified in vitro and reintroduced in the parent strain via allele exchange using suicide vector pCVD442. The mutant strain thus generated (DM34) had its entire fim cluster removed. Its pathogenic properties were compared to those of the parent strain in an experimental reproduction of avain colibacillosis in 15-day-old chickens, after primary infection with infectious bronchitis virus followed by intratracheal inoculation of the challenge strain. In specific-pathogen-free (SPF) animals, mutant DM34 was less pathogenic than the parent strain and colonized the lungs of infected animals to a lower level. In germ-free chickens, although DM34 was less pathogenic than MT78 according to the differences in weight gains, it colonized the trachea, lungs and internal organs to the same extent as MT78. Our results suggest that, whereas type 1 fimbriae are not strictly required in colonization of the respiratory tract of germ-free chickens, they might be important in establishing a bacterial population in the lungs of SPF animals. The difference regularly observed in weight gains between mutant- and wild-type-inoculated chickens reflects a decreased pathogenicity of the fim- mutant. However, the isolation of E. coli in target organs and the observation of colibacillosis symptoms and lesions in mutant-inoculated chickens suggest that type 1 fimbriae by themselves play a limited role in pathogenicity.

Localization of the in vivo expression of P and F1 fimbriae in chickens experimentally inoculated with pathogenic Escherichia coli.

Escherichia coli causing septicemia in poultry often possess F1 (type 1) and/or P fimbriae which may be involved in bacterial colonization and infection. To investigate the expression of these fimbriae in vivo, two pathogenic E. coli strains with different fimbrial profiles, TK3 (fim+/pap+) and MT78 (fim+/pap-), were administered to 2-week-old chickens by either the intratracheal or caudal thoracic air sac inoculation route. Antibodies specific for native F1 fimbriae were detected by ELISA and immunodot in the serum of chickens inoculated with either strain MT78 or strain TK3, irrespective of the route of inoculation. Antibodies specific for P fimbriae of serotype F11 were detected by ELISA and immunoblotting in the serum of chickens inoculated by either route with strain TK3. F1, but not P fimbriae, were expressed by bacteria colonizing the trachea of chickens inoculated by the air sac route with strain MT78 or TK3, as demonstrated by examination of frozen tissue sections using immunofluorescence. F1 fimbriae were also expressed by bacteria colonizing the air sacs and lungs, but not by bacteria in the blood or other internal organs, of chickens inoculated with either strain. P fimbriae were expressed by bacteria colonizing the air sacs, lungs, kidney, blood, and pericardial fluid, but not by bacteria colonizing the trachea, of chickens inoculated with strain TK3. Fimbriae-like structures were observed by electron microscopy on bacteria adhering to the epithelial cells of the air sacs of chickens inoculated with strain TK3. These results demonstrate that both strains MT78 and TK3 undergo in vivo phase variation with respect to their fimbrial profiles and site of bacterial colonization in different organs of infected chickens and suggest that F1 fimbriae are important for initial bacterial colonization of the upper respiratory tract whereas P fimbriae are important for later stages of the infection.

Analysis of the fim cluster of an avian O2 strain of Escherichia coli: serogroup-specific sites within fimA and nucleotide sequence of fimI.

Escherichia coli MT78, an avian pathogenic strain of serogroup 02, produces a variant form of type 1 fimbriae with distinct antigenic properties and apparent mol. wt of the major subunit. The fim gene cluster of strain MT78 was cloned and its sequence was determined in a region spanning upstream of fimB to the beginning of fimD. Whereas most genes were well conserved relative to fim genes previously described, comparison of the fimA gene from strain MT78 with homologous sequences from other strains of E. coli and Klebsiella pneumoniae revealed that most differences were clustered in four well defined regions. A PCR assay, based upon these variable sequences, allowed amplification of a fragment of gene fimA which is specific for most 02 strains. In addition, the sequence of the previously uncharacterised gene fimI, which is located between genes fimA and fimC, was determined.

Results of passive and active immunization directed against ferric aerobactin in experimental enterobacterial infections in mice and chickens.

Production of aerobactin has been reported to be a virulence factor in members of the family Enterobacteriaceae. To investigate the protection afforded by humoral immunity directed towards aerobactin in infectious diseases caused by aerobactin-producing strains, we tested the efficacy of mAbAERO1, a murine monoclonal antibody directed to ferric aerobactin, which, in vitro, was found to impair the growth of aerobactin-dependent strains of Enterobacteriaceae under iron-limited conditions. The mortality of mice experimentally infected with the aerobactin-producing strains Escherichia coli V2019 (LD50 = 3.5 x 10(5) CFU/mice) or Klebsiella pneumoniae Caroli (LD50 = 1.3 CFU/mice) was not reduced when 1 mg of mAbAERO1 was injected intravenously 1 h before or 1 h after bacterial challenge. Nor was mortality reduced after challenge with either E. coli V2019 or K. pneumoniae Caroli, even though the active immunization of mice with purified FeAero (ferric aerobactin) conjugated with thyroglobulin as followed by a rise in systemic anti-FeAero antibodies. Lastly, chicks born of hens immunized with FeAero showed evidence of antibody transmission towards FeAero, but were not protected when challenged with E. coli MT78, an aerobactin-producing strain highly virulent for chickens. Therefore, under the experimental conditions tested, humoral immunity against aerobactin appeared to play only a minor role in protection against infections caused by aerobactin-producing members of the family Enterobacteriaceae. However, other experimental models should be tested to confirm these observations.

Immunological variants of the aerobactin-cloacin DF13 outer membrane protein receptor IutA among enteric bacteria.

Mouse monoclonal antibodies (MAbs) were generated against a 76-kDa IutA receptor of pathogenic avian Escherichia coli 15972. Six of the eight IutA-specific MAbs isolated (AB1 to AB6) were shown to be directed toward membrane-exposed conformational epitopes, although they did not interfere with the uptake of ferric aerobactin and cloacin DF13 as assessed by competition experiments with purified ligands. The two remaining IutA MAbs (AB9 and AB10) recognized linear epitopes buried in the IutA molecule. The panel of IutA MAbs was used to characterize IutA variants occurring in strains of E. coli, Klebsiella pneumoniae, Enterobacter spp., and Shigella spp., resulting in the identification of four immunological groups of IutAs. MAb AB9 defined an epitope conserved in all IutA variants. In addition, the panel of IutA MAbs served to identify the presence of IutA in wild-type bacteria grown in the presence of diphenylamine to reduce the expression of O-specific polysaccharide.

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.

Clonal relationships and variation in virulence among Escherichia coli strains of avian origin.

Average genetic relatedness among 44 Escherichia coli strains of serotypes O1, O2, and O78 isolated mainly from birds with colibacillosis or swollen-head syndrome from France or Saudi Arabia was estimated based on allelic variation detected by multilocus enzyme electrophoresis. For 20 enzyme-encoding loci, we resolved 2.8 alleles per locus and distinguished 17 electrophoretic types (ETs) that were used to mark naturally occurring cell lineages or clones. On average, ETs differed at 37% of their loci. Forty-eight percent of the isolates represent three ETs, two of which belong to previously defined complexes of clones identified in avian disease in North America and Europe. Virulence of strains, assessed in experimental infections of day-old chicks, showed little variation among isolates of a clone, but was significantly variable among isolates of different clone complexes. These findings add support to the evidence that a majority of avian isolates that cause colibacillosis belong to a few cosmopolitan pathogenic clones and indicate a substantial between-clone component of pathogenicity.

Serological conservation and location of the adhesin of avian Escherichia coli type 1 fimbriae.

By inoculation of mice with purified type 1-like fimbriae isolated from an avian Escherichia coli strain, a monoclonal antibody (mAb G5) was obtained. mAb G5 reacted in an enzyme-linked immunosorbent assay (ELISA) with type 1-like and type 1A fimbriae differing in the molecular masses of their major fimbrial subunit and isolated from several avian E. coli strains. The specificity of mAb G5 for type 1 fimbriae was assessed in a whole bacteria ELISA with 16 reference E. coli strains expressing different types of fimbriae. Immunoblotting experiments showed that mAb G5 recognized the 29 kDa minor component of reference type 1A fimbriae which has been identified as the adhesin. mAb G5 also recognized the 29 kDa component of type 1-like and type 1A fimbriae expressed by avian E. coli strains, suggesting that the adhesin is antigenically conserved among these fimbriae. Immunoelectron microscopic studies gave evidence that the adhesin could be located mainly at the tip or both at the tip and along the fimbriae, depending on the strain.

Surface antigens from Escherichia coli O2 and O78 strains of avian origin.

Fimbriae from O2 and O78 virulent strains of avian Escherichia coli were compared with type 1A fimbriae with regard to the apparent molecular weights of their subunits and their antigenic relationships. Under static broth culture conditions, most O78 strains expressed fimbriae closely related to those of type 1A. Under the same culture conditions, another type of fimbriae, sharing some common properties with type 1A fimbriae, was observed only on O2 strains; however, these fimbriae differed from type 1A fimbriae in the apparent molecular weights of their subunits and in the expression of specific epitopes. They were called type 1-like fimbriae. Homologies in lipopolysaccharide and outer membrane protein profiles were also demonstrated among the strains expressing type 1-like fimbriae, which suggests the existence of a clonal relationship among O2:K1 avian E. coli strains. The O78 strains studied did not appear to be clonally related.