A new multidrug-resistant enterotoxigenic Escherichia coli pulsed-field gel electrophoresis cluster associated with enrofloxacin non-susceptibility in diseased pigs.

AIMS: To describe the temporal trends in Escherichia coli pathotypes and antimicrobial resistance detected in isolates from diseased-pig cases submitted to the EcL from 2008 to 2016, in Quebec, Canada, and to investigate the presence of spatiotemporal and phylogenetic clusters. METHODS AND RESULTS: Detection of 12 genes coding for virulence factors in pathogenic E. coli in pigs by PCR and antimicrobial resistance standard disc diffusion assay were performed. Demographic and clinical data were entered in the Animal Pathogenic and Zoonotic E. coli (APZEC) database. ETEC:F4 was the most prevalent pathovirotype among the 3773 cases submitted. The LT:STb:F4 virotype was predominant until 2014, then was overtaken by the LT:STb:STa:F4 virotype. More than 90% of the ETEC:F4 isolates were multidrug resistant. A spatiotemporal cluster of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin was detected between 4/2015 and 9/2016. Pulsed-field gel electrophoresis analysis of 137 ETEC:F4 isolates revealed the presence of a cluster composed mainly of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin. CONCLUSIONS: The APZEC database was useful to highlight temporal trends in E. coli pathotypes. A high-risk ETEC:F4 clone might disseminate in the pig population in Quebec since 2015. SIGNIFICANCE AND IMPACT OF THE STUDY: Surveillance is crucial to identify new clones and develop control strategies.

Effect of Cessation of Ceftiofur and Substitution with Lincomycin-Spectinomycin on Extended-Spectrum-ß-Lactamase/AmpC Genes and Multidrug Resistance in Escherichia coli from a Canadian Broiler Production Pyramid.

Ceftiofur, a third-generation cephalosporin antimicrobial, was used in Canadian hatcheries for many years to prevent early mortality in chicks, leading to a high prevalence of cephalosporin resistance in Escherichia coli in chickens. Preventive use of ceftiofur in hatcheries ceased in 2014. We examined the effect of ceftiofur cessation (n = 40 flocks with ceftiofur and n = 28 flocks without antimicrobial at hatchery) and its replacement with an antimicrobial combination, lincomycin-spectinomycin (n = 32), at the hatchery on the proportion of samples with E. coli positive for extended-spectrum-ß-lactamase (ESBL) and AmpC ß-lactamase-related genes, and on the multidrug resistance profiles of ESBL/AmpC-positive E. coli in broilers and their associated breeders (n = 46 samples), at 1 year postcessation. For indicator E. coli from nonenriched media, a significant decrease postcessation in the proportion of samples harboring E. coli isolates positive for blaCMY-2 and/or blaCTX-M was observed. In contrast, following enrichment in medium containing ceftriaxone (1 mg/liter) to facilitate recovery of ESBL/AmpC ß-lactamase-producing E. coli colonies, both pre- and postcessation, 99% of the samples harbored E. coli positive for blaCMY-2 or blaCTX-M Among the 15 tested antimicrobial agents, flocks receiving lincomycin-spectinomycin after cessation of ceftiofur showed a significantly greater nonsusceptibility to aminoglycosides, folate inhibitors, phenicols, and tetracyclines and a greater proportion of possible extensively drug-resistant E. coli than those receiving ceftiofur or no antimicrobial at hatchery. This study clearly demonstrates an initial decrease in ESBL/AmpC-positive E. coli following the cessation of ceftiofur in the hatchery but an increase in antimicrobial non-ß-lactam resistance of ESBL/AmpC-positive E. coli following replacement with lincomycin-spectinomycin.IMPORTANCE Antimicrobial resistance is a global problem. The antimicrobial ceftiofur has been used worldwide for disease prevention in poultry production, resulting in a greatly increased resistance to this antimicrobial important in poultry and human medicine. Our study examined the impact of ceftiofur cessation and its replacement with the antimicrobial combination lincomycin-spectinomycin, a common practice in the industry. Our study demonstrated a decrease in ceftiofur resistance after the cessation of ceftiofur use, although the resistance genes remain ubiquitous in all phases of poultry production, showing that poultry remains a reservoir for ceftiofur resistance and requiring continued vigilance. We also observed a decrease in multidrug resistance involving different antimicrobial classes after cessation of ceftiofur but an increase following use of lincomycin-spectinomycin, indicating that this antimicrobial use should be questioned. Reduced resistance to ceftiofur in poultry may translate to better treatment efficacy, decreased morbidity/mortality, and enhanced food safety for humans.

Efficacy of a single oral dose of a live bivalent E. coli vaccine against post-weaning diarrhea due to F4 and F18-positive enterotoxigenic E. coli.

F4- and F18-positive enterotoxigenic E. coli strains (F4-ETEC and F18-ETEC) are important causes of post-weaning diarrhea (PWD) in pigs. F4 (antigenic variant ac) and F18 (ab and ac) fimbriae are major antigens that play an important role in the early stages of infection. Herein, the efficacy of a live oral vaccine consisting of two non-pathogenic E. coli strains, one F4ac- and one F18ac-positive, was evaluated using F4ac-ETEC and F18ab-ETEC challenge models. A randomized, masked, placebo-controlled, block design, parallel-group confirmatory study with two different vaccination-challenge intervals (7 and 21 days) was conducted for each challenge model. The vaccine was administered in one dose, to ≥18-day-old piglets via drinking water. Efficacy was assessed by evaluating diarrhea, clinical observations, weight gain and fecal shedding of F4-ETEC or F18-ETEC. Anti-F4 and anti-F18 immunoglobulins in blood were measured. The vaccination resulted in significant reductions in clinical PWD and fecal shedding of F4-ETEC and F18-ETEC after the 7- and 21-day-post-vaccination heterologous challenges, except for after the 21-day-post-vaccination F4-ETEC challenge, when the clinical PWD was too mild to demonstrate efficacy. A significant reduction of mortality and weight loss by vaccination were observed following the F18-ETEC challenge. The 7-day protection was associated with induction of anti-F4 and anti-F18 IgM, whereas the 21-day protection was mainly associated with anti-F4 and anti-F18 IgA. The 7-day onset and 21-day duration of protection induced by this vaccine administered once in drinking water to pigs of at least 18days of age were confirmed by protection against F4-ETEC and F18-ETEC, and induction of F4 and F18-specific immunity. Cross protection of the vaccine against F18ab-E. coli was demonstrated for both the 7- and 21-day F18-ETEC challenges.

Circulating of CMY-2 ß-lactamase gene in weaned pigs and their environment in a commercial farm and the effect of feed supplementation with a clay mineral.

AIMS: To investigate the mechanisms leading to an increase in the prevalence of blaCMY -2 conferring resistance to ceftiofur in pigs receiving a feed medicated with chlortetracycline and penicillin, and to examine the effect of supplementation with a clay mineral on this phenomenon. METHODS AND RESULTS: In 138 blaCMY -2 -positive Escherichia coli isolates from faeces of pigs receiving feed supplemented or not with 2% clinoptilolite, from day 2 to day 28 after weaning, isolates from the two groups differed significantly with respect to their phylogenetic group: phylotype A predominated in the supplemented group, whereas phylotypes B1 and D predominated in the control group, as determined by PCR. In 36 representative isolates, pulsed-field gel electrophoresis and antimicrobial susceptibility testing revealed that the blaCMY -2 -positive E. coli isolates were polyclonal with diverse antimicrobial resistance patterns and blaCMY -2 -carrying plasmids of incompatibility (Inc) groups, A/C, I1 and ColE were observed in transformants as detected by PCR. Enterobacter cloacae possessing blaCMY -2 -carrying IncA/C plasmids were found in the pens before introduction of this batch of pigs. The blaCMY -2 -positive E. coli isolates were more clonally diverse in the control group than the supplemented group. CONCLUSIONS: The blaCMY -2 gene appears to have spread both horizontally and clonally in this batch of pigs and may have spread from previous batches of pigs via plasmids carried by Ent. cloacae and expanded in animals of the present batch in the presence of the selection pressure due to administration of chlortetracycline and penicillin in the feed. Feed supplementation may have an effect on clonal diversity of blaCMY -2 -positive isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of improved hygiene measures, decreased administration of certain antimicrobials on farm and feed supplementation with certain ingredients may limit antimicrobial resistance spread between and within batches of animals.

Impact of mash feeding versus pellets on propionic/butyric acid levels and on total load in the gastrointestinal tract of growing pigs.

Feed characteristics may influence the bacterial community composition and metabolic activities in the pig gastrointestinal tract, known to be associated with positive effects on the gut. Use of mash feed is associated with reduced excretion, but little is known of its effect on the population or of the mechanism of action. Our objectives were to assess the effect of feed texture combined with feed particle size on VFA profiles and levels, total count, and the presence of genes encoding virulence factors of pathogenic strains in the digestive tract along with their impact on pig performance of fattening pigs. Pigs ( = 840) on a commercial farm received mash or pellet diets of different particle sizes during the fattening period. Caecal and colon contents from 164 pigs were sampled at the slaughterhouse for enumeration of by quantitative PCR (qPCR) and for VFA quantification by capillary gas chromatography. The gene was used to enumerate total . Improved pig performances associated with pellet texture and a 500-µm size were observed. Caecal ( = 0.02) and colon ( < 0.01) propionic acid concentrations were lower for pigs receiving pellet rather than mash feed. Similarly, caecal ( = 0.01) and colon ( < 0.001) butyric acid concentrations were also lower for pigs receiving pellet rather than mash feed, as determined by capillary gas chromatography. Moreover, caecal ( = 0.03) and colon ( < 0.001) butyric acid concentrations were higher for pigs receiving a feed with a 1,250-µm particle size rather than a 500-µm particle size. On the other hand, total caecal and colon levels were higher for pigs receiving pellet feed than for those receiving mash feed. For total enumeration, caecal ( < 0.01) and colon ( < 0.01) gene copies were higher for pigs receiving pellet rather than mash feed. No effect of particle size on fatty acid concentrations or on numbers was observed. Virulence gene quantification revealed no trend. Taken together, results showed that mash feed is associated with lower growth performance but with favorable intestinal changes linked to VFA levels and reduction in the intestine.

Relationship between Escherichia coli virulence factors and postpartum metritis in dairy cows.

The objectives of this study were to report the prevalence of Escherichia coli and Trueperella pyogenes in the uterus of postpartum dairy cows before the onset of postpartum metritis (PPM) and to quantify their association with subsequent occurrence of PPM, to quantify the association between the presence of genes encoding E. coli virulence factors (VF) and PPM, and to determine the accuracy of using early postpartum uterine bacteriology results (bacteria and VF) to identify cows at risk of PPM. A prospective cohort study was conducted on 3 commercial dairy farms. Uterine swabs were collected from 371 Holstein dairy cows (3 commercial herds) at 1 to 7d in milk and submitted to the laboratory for identification of E. coli, T. pyogenes, and E. coli VF. A total of 40 VF were tested using the radioactive probe hybridization method. Postpartum metritis was defined as the presence of a fetid watery red-brown uterine discharge, associated with fever (rectal temperature >39.5°C), and systemic signs of illness (dullness, reduced appetite, and milk production). Surveillance of PPM was done by trained farmers blinded to laboratory results and cows were followed until 21d in milk. Statistical analyses were conducted using 2×2 tables and mixed logistical regression models. Prevalences of E. coli, T. pyogenes, and PPM were 42, 34, and 15%, respectively. A total of 32 VF were found in E. coli isolates. Most prevalent VF were extraintestinal pathogenic genes such as fimH (89%), hlyE (87%), and iss (70%). Cows positive for intrauterine E. coli were 3.2 times more likely to have subsequent PPM compared with bacteriologically negative cows. Cows with VF hra1 in their uterus were 2.7 times more likely to have PPM than cows positive for E. coli and negative for hra1 and 5.9 times more likely than bacteriologically negative cows. Cows with VF kpsMTII in their uterus were 3.2 times more likely to have PPM than cows positive for E. coli and negative for kpsMTII and 6.2 times more likely than bacteriologically negative cows. Using E. coli, hra1, and kpsMTII as predictors for subsequent PPM, positive predictive values were 23, 31, and 42%, respectively, whereas the negative predictive values were 91, 80, and 78%, respectively. Overall, these results showed that E. coli and some VF were associated with PPM.

Antimicrobial resistance and virulence gene profiles in multi-drug resistant enterotoxigenic Escherichia coli isolated from pigs with post-weaning diarrhoea.

This study aimed to characterize antimicrobial resistance and virulence genes in multi-drug resistant enterotoxigenic Escherichia coli (ETEC) isolates (n=117) collected from porcine post-weaning diarrhoea cases in Australia (1999-2005). Isolates were serotyped, antibiogram-phenotyped for 12 antimicrobial agents and genotyped by PCR for 30 plasmid-mediated antimicrobial resistance genes (ARGs), 22 intestinal and 38 extraintestinal E. coli virulence genes (VGs). Nine serogroups were identified, the most prevalent being O149 (46.2%), O141 (11.2%) and Ont (31.6%). None of the isolates showed resistance to ceftiofur or enrofloxacin and 9.4% were resistant to florfenicol. No corresponding extended-spectrum/AmpC ß-lactamase, fluoroquinolone or floR ARGs were detected. An antimicrobial resistance index (ARI) was calculated from the combined data with a weighting for each antimicrobial agent dependent upon its significance to human health. Serogroup O141 isolates had a significantly higher ARI due to an elevated prevalence of aminoglycoside ARGs and possession of more virulence genes (VGs), including ExPEC or EHEC adhesins (bmaE, sfa/focDE, fimH, ihA) in toxin-producing strains that lacked the normally associated F4 and F18 fimbriae. Few associations between ARGs and VGs were apparent, apart from tetC, sfa/focDE and ompT which, for a sub-set of O141 isolates, suggest possible plasmid acquisition from ExPEC. The multi-drug resistant ETEC ARG/VG profiles indicate a high probability of considerable strain and plasmid diversity, reflecting various selection pressures at the individual farm level rather than emergence and lateral spread of MDR resistant/virulent clones.

Administration of Pediococcus acidilactici or Saccharomyces cerevisiae boulardii modulates development of porcine mucosal immunity and reduces intestinal bacterial translocation after Escherichia coli challenge.

In this study, the influence of the probiotics, Pediococcus acidilactici (PA) and Saccharomyces cerevisiae boulardii (SCB), on intestinal immune traits and resistance to enterotoxigenic Escherichia coli (ETEC) infection was evaluated in pigs. Two weeks before farrowing, 30 sows and their future litters were allocated to the following treatments: 1) control group without antibiotic or probiotic treatment (CTRL), 2) control with antibiotic (tiamulin) added to weanling feed (ABT), or litters treated with 3) PA, 4) SCB, or 5) PA+SCB from 24 h after birth. During lactation, PA, SCB, or PA+SCB were given to piglets 3 times a week by gavage. After weaning at 21 d of age, probiotics or ABT were added to the diet. Four pigs per litter were chosen to evaluate performance and blood concentrations of folic acid and vitamin B(12). Three of these were orally challenged with an ETEC strain on d 49 to 51 and killed on d 52. Three piglets from the rest of the litter were slaughtered on d 18 and 3 others on d 24. Blood, ileum, and mesenteric lymph node (MLN) samples were taken to characterize leukocyte populations, determine IgA concentrations in ileal flushes, and evaluate bacterial translocation in MLN. No treatment effect on postweaning performance and on blood concentrations of folic acid and vitamin B(12) was observed. In the ileum, the percentage of CD4(-)CD8(+low) T cells was greater (P = 0.05) in 18-d-old nursed piglets treated with PA than in those of the CTRL and PA+SCB groups. In the MLN, the percentage of CD8(+) T cells was not affected by any of the treatments at d 18 and 24 but decreased (P = 0.006) after weaning. In the blood, CD8(+) T cells were not affected by treatments or weaning. After the ETEC challenge (d 52), bacterial translocation to MLN was reduced (P = 0.05) in pigs treated with PA, SCB, PA+SCB, or ABT compared with CTRL. No treatment effect was observed on blood leukocyte populations after ETEC challenge, although a time effect (d 42 vs. 52) indicated that blood CD4(+) and gammadelta-T lymphocytes were increased (P < 0.05) on d 52 compared with d 42, whereas CD4(-)CD8(+low) T lymphocytes and monocytes were markedly reduced (P < 0.01). Finally, the IgA concentration in ileal flushes collected on d 42 and 52 was greater in SCB and CTRL piglets than in ABT and PA piglets. In conclusion, probiotics may have the potential to modulate establishment of lymphocyte populations and IgA secretion in the gut and to reduce bacterial translocation to MLN after ETEC infection.

Improvement of adjuvant systems to obtain a cost-effective production of high levels of specific IgY.

Incomplete Freund's adjuvant (IFA) is used as standard adjuvant for the production of specific antibodies. In this study, we evaluated the ability of supplementation of IFA with 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or C-phosphate-guanosine-oligodeoxynucleotide (CpG-ODN) to enhance the quantity of specific IgY found in the eggs of hyperimmunized laying hens. In this comparative study, the fimbrial adhesin F4 of porcine enterotoxigenic Escherichia coli was used as prototype immunogen. Hens of 3 groups received by i.m. injection 20 microg of purified F4 adhesin emulsified with 1 of the following adjuvants: 0.5 mL of IFA alone (F4-IFA group), 0.5 mL of IFA supplemented with 285.6 ng of 1alpha,25(OH)(2)D(3) (F4-IFA-D(3) group), or 0.5 mL of IFA supplemented with 10 microg of CpG-ODN (F4-IFA-CpG group). Hens of 2 control groups received PBS or purified F4 alone. Immunization was repeated after 2 and 5 or 7 wk. Eggs were collected at 3- to 4-d intervals from preimmunization to d 79, and whole eggs were tested to measure the quantity of anti-F4 IgY by a standardized indirect ELISA. The quantity of specific anti-F4 IgY present in eggs from immunized hens of the F4-IFA group increased from d 13 to 79, corresponding to the end of the experiment. The values for this group at each time were considered as 100%. Results obtained for the other adjuvants were expressed in relation to this reference method. Supplementation of IFA with 1alpha,25(OH)(2)D(3) did not result in any enhancement of the quantity of anti-F4 IgY present in the eggs. On the other hand, supplementation of IFA with CpG-ODN resulted in an enhancement of yield up to 942% of the F4-specific antibody response. Moreover, the use of CpG-ODN is a cost-effective and ethical refinement for the production of specific antibodies, permitting a reduction in the number of immunizations needed. In conclusion, this study provides strong evidence for the use of IFA supplemented with CpG-ODN rather than IFA alone for the production of high levels of specific antibody in laying hens.

Escherichia coli: on-farm contamination of animals.

Escherichia coli is one of the main inhabitants of the intestinal tract of most mammalian species, including humans, and birds. Shiga toxin-producing E. coli (STEC), also called verotoxinogenic E. coli, usually do not cause disease in animals but may cause watery diarrhoea, haemorrhagic colitis, and/or haemolytic uraemic syndrome in humans. Zoonotic STEC include the O157:H7 strains and, with increasing frequency, certain non-O157 strains. The importance of non-O157 zoonotic strains is probably underestimated as they have been less well characterised and are more difficult to detect in samples than O157:H7. Another large subset of STEC strains has been isolated from animals but has not, at the present time, been associated with disease in animals or humans. Cattle and other ruminants are the most important reservoir of zoonotic STEC, which are transmitted to humans through the ingestion of foods or water contaminated with animal faeces, or through direct contact with the infected animals or their environment. The main sources of STEC infection of cattle on-farm are the drinking water, the feed, and the immediate environment of the animal. Risk factors that have been identified for infection of animals with O157 STEC include age, weaning, movement of the animals, season, feed composition, and the ability of the bacteria to persist in the environment. On-farm control of the zoonotic risk of human infection with STEC should primarily target the main source of contamination: the animal reservoir. Various strategies to reduce intestinal colonisation of cattle by zoonotic STEC have been tried with varying results, including vaccination, treatment with probiotics, such as direct-fed microbials or competitive exclusion, administration of bacteriophages, and modification of the diet.

Virulence factors in Escherichia coli isolated from the blood of bacteremic neonatal calves.

Twenty-five Escherichia coli isolates from the blood of critically ill bacteremic calves sampled in two separate studies on a calf-rearing farm housing over 15,000 calves, in the San Joaquin Valley, California were studied. Isolates were characterized for O serogroups and for pathotypes as determined by the presence of specific virulence factors including heat-labile enterotoxin (LT), heat-stable enterotoxins a and b (STa, STb), verotoxins 1 and 2 (VT1, VT2), cytotoxic necrotizing factor (CNF), aerobactin, intimin Eae and P, F17 and CS31A fimbrial adhesins, and resistance to bactericidal effects of serum. These isolates constituted a heterogeneous group. However, isolates were mostly aerobactin positive and often resistant to the bactericidal effects of serum. Isolates of pathotypes O78 (n=6), O119:CS31a (n=3), and P positive but O non-typeable (n=3) were associated with a high mortality rate. The remaining isolates belonged to diverse pathotypes, often possessing the adhesins P, F17, CS31A and Eae but belonging to O serogroups other than O78 and O119, and were less frequently associated with mortality. Although no virulence factor common to all isolates was identified, the capacity to use iron by the presence of aerobactin which is important to the capture of iron was a predominant factor. Moreover, certain pathotypes appear to be associated with primary colisepticemia whereas other pathotypes may cause a bacteremia without necessarily leading to septicemia.

Phenotypic and genotypic characterization of Escherichia coli verotoxin-producing isolates from humans and pigs.

The aim of this study was to characterize verotoxin-producing Escherichia coli (VTEC) isolates obtained from humans and pigs in the same geographic areas and during the same period of time in order to determine whether porcine VTEC isolates could be related to human cases of diarrhea and also to detect the presence of virulence factors in these isolates. From 1,352 human and 620 porcine fecal samples, 11 human and 18 porcine verotoxin-positive isolates were obtained by the VT immunoblot or the individual colony testing technique. In addition, 52 porcine VTEC strains isolated from diseased pigs at the Faculté de médecine vétérinaire during the same period or from fecal samples collected previously isolated at slaughterhouses were characterized in this study. Antimicrobial resistance profiles were different between human and porcine isolates. In general, the serotypes observed in the two groups were different. No porcine isolate was of serotype O157:H7; however, one isolate was O91:NM, a serotype that has been associated with hemorrhagic colitis in humans. Also, one serotype (O8:H19) was found in isolates from both species; however, the O8:H19 isolates of the two groups were of different pathotypes. The pathotypes observed in the human and porcine isolates were different, with the exception of VT2vx-positive isolates; the serotypes of these isolates from the two groups were nevertheless different. Pulsed-field gel electrophoresis analysis indicated no relatedness between the human and porcine isolates. In conclusion, these results suggest that the porcine and human isolates of the present study were not genetically related. Most porcine VTEC isolates did not possess known virulence factors required to infect humans. However, certain non-O157:H7 porcine VTECs may potentially infect humans.

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.

Presence of the LEE (locus of enterocyte effacement) in pig attaching and effacing Escherichia coli and characterization of eae, espA, espB and espD genes of PEPEC (pig EPEC) strain 1390.

In the present study, attaching and effacing Escherichia coli (AEEC) O45 isolates from post-weaning pigs with diarrhoea were examined for the presence of the LEE (locus of enterocyte effacement) using various DNA probes derived from the LEE of human enteropathogenic E. coli (EPEC) strain E2348/69. The LEE fragment was conserved among the eae -positive pig isolates. The attaching and effacing activity of PEPEC (pig EPEC) O45 isolates is highly correlated with the presence of the LEE. Nevertheless, for some PEPEC isolates, the insertion site of the LEE is different or has diverged during evolution. The presence of the LEE fragment in PEPEC isolates provides further evidence that the LEE region is conserved among AEEC of different animal origins. In addition, the nucleotide sequence of the region containing the eae gene and esp genes of a pig AEEC isolate, strain 1390, was determined. Among examined Eae proteins, Eae of strain 1390 showed the highest similarity with Eae belonging to the beta intimin group such as the Eae of rabbit AEEC. Moreover, all pig strains that produced attaching and effacing lesions in piglets and pig ileal explants belonged to the beta intimin group. The deduced amino acid sequences of the EspA, EspB and EspD proteins of strain 1390 showed particularly strong homology to those of AEEC strains presenting a beta intimin allele. Thus, pig AEEC possess the LEE sequences, and for the strain 1390, sequences of the eae and esp regions are related to those of other AEEC, in particular, strains presenting a beta intimin allele, such as the rabbit AEEC.

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.

Comparison of necrotoxigenic Escherichia coli isolates from farm animals and from humans.

Necrotoxigenic Escherichia coli (NTEC) isolated from animals and humans can belong to the same serogroups/types and produce or carry the genes coding for fimbrial and afimbrial adhesins of the same family, P, S, F17, and/or AFA, raising the question of a potential zoonotic source of human infection. The main purpose of this study was to compare 239 NTEC1 strains (45 from cattle, 65 from humans and 129 from piglets) and 98 NTEC2 strains from cattle, using a uniform and standardized typing scheme. The O serogroups and the biotypes recognized amongst NTEC1 and NTEC2 strains were quite varied, although some were more frequently observed (serogroups O2, O4, O6, O8, O18, O78, and O83 and biotypes 1, 2, 5, 6, and 9). Hybridization, results with gene probes for the P family (PAP probe), S family (SFA probe), AFA family (AFA probe), F17 family (F17 probe) of fimbrial and afimbrial adhesins, could differentiate most NTEC1 strains, which are PAP-, SFA- and/or AFA-positive, from NTEC2 strains, which are mainly F17- and/or AFA-positive, but were of no help in differentiating between NTEC1 strains from cattle, humans, and piglets. All but seven (98%) NTEC1 and NTEC2 strains were serum resistant, 199 (59%) produced an aerobactin, and colicin (I, V, or unidentified) was produced by 22-34% of them. On the other hand, more than 90% of the NTEC1 strains were haemolytic on sheep blood agar compared with only 40% of the NTEC2 strains. Production of a classical haemolysin, active on sheep erythrocytes, and hybridization with the PAP probe were associated in a majority of NTEC1 strains (63-81%), but very rarely in NTEC2 strains (3%). Production of enterohaemolysin and hybridization with the PAP probe were much less frequently associated in NTEC strains (1-9%). It was thus possible neither to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotizing Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additionnal methods.

F165(1) fimbriae of the P fimbrial family inhibit the oxidative response of porcine neutrophils.

The F165(1) fimbrial system has been associated with the resistance of Escherichia coli O115:K"V165" to phagocytic killing by porcine polymorphonuclear leukocytes (PMNLs). One mechanism of this resistance seemed to be inhibition of the oxidative response as observed following induction of PMNLs by phorbol myristate acetate (PMA) and treatment with bacteria possessing the F165(1) fimbriae. In order to confirm whether or not the F165(1) fimbriae are involved in this inhibition, we evaluated the effect of F165(1)-positive strains (a pathogenic wild-type strain 5131, and a recombinant strain HB101(pCJ7)) or an F165(1)-negative strain HB101 (used as negative control) on the oxidative response of porcine neutrophils (pNs) stimulated with PMA. Incubation of pNs with pathogenic E. coli strain 5131 resulted in significant inhibition of the oxidative response as compared to that observed for pNs incubated without bacteria, as assessed by hydrogen peroxide (H2O2) and superoxide anion (O2-) release from the phagocytes, and by the chemiluminescence assay. Similarly, incubation of pNs with the F165(1)-producing cloned strain HB101(pCJ7) resulted in significant inhibition of the pN oxidative response as compared to that observed for pNs incubated without bacteria or with strain HB101. In contrast, addition of purified F165(1) fimbriae to the pNs had no effect on the oxidative response.

Characterization of the risk to human health of pathogenic Escherichia coli isolates from chicken carcasses.

The purpose of this study was to evaluate the risk for human health associated with pathogenic Escherichia coli isolated from airsacculitis and cellulitis in chickens, by comparing the genotypic and phenotypic profiles of avian E. coli isolates and E. coli strains isolated from sick humans during the same period and in the same geographical area as the avian isolates. A total of 96 isolates and 46 isolates from lesions of cellulitis and airsacculitis, respectively, were obtained. Isolates from the backs of some of the affected and healthy birds and 91 intestinal and extraintestinal isolates from humans with diarrhea, urinary tract infections, or septicemia were examined. The frequency of antimicrobial resistance was in general higher in the avian than in the human isolates. VT1-VT2-Eae and VT2-Eae, pathotypes associated with hemolytic and uremic syndrome and bloody diarrhea in humans, were the most frequently encountered pathotypes in human intestinal isolates but were not recovered from the avian isolates. Aero-Pap-TSH and Aero-TSH were the most frequently encountered pathotypes in avian isolates but were rarely observed in human isolates. No avian isolate was of serogroup O157, whereas many human isolates belonged to this O group. O78 and O2 were the most frequently observed O groups in avian isolates but were rarely found in human isolates. Only two avian isolates demonstrated possible relatedness to human isolates based on pulsed-field gel electrophoresis profiles, but they belonged to different pathotypes. Our results suggest that avian isolates recovered from cellulitis and air sacullitis possess very few of the attributes required to cause diseases in humans. It is also concluded that isolates from cellulitis and airsacculitis do not represent a greater hazard than isolates from the back of healthy birds.

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.