Martine Boulianne

Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic E. coli

Extraintestinal pathogenic Escherichia coli (ExPEC) are an important cause of urinary tract infections, neonatal meningitis and septicaemia in humans. Animals are recognized as a reservoir for human intestinal pathogenic E. coli, but whether animals are a source for human ExPEC is still a matter of debate. Pathologies caused by ExPEC are reported for many farm animals, especially for poultry, in which colibacillosis is responsible for huge losses within broiler chickens. Cases are also reported for companion animals. Commensal E. coli strains potentially carrying virulence factors involved in the development of human pathologies also colonize the intestinal tract of animals. This review focuses on the recent evidence of the zoonotic potential of ExPEC from animal origin and their potential direct or indirect transmission from animals to humans. As antimicrobials are commonly used for livestock production, infections due to antimicrobial-resistant ExPEC transferred from animals to humans could be even more difficult to treat. These findings, combined with the economic impact of ExPEC in the animal production industry, demonstrate the need for adapted measures to limit the prevalence of ExPEC in animal reservoirs while reducing the use of antimicrobials as much as possible.

Virulence mechanisms of avian fimbriated Escherichia coli in experimentally inoculated chickens

Virulence mechanisms of avian pathogenic Escherichia coli were investigated by inoculating commercial broiler chickens via the left caudal thoracic air sac with three highly pathogenic and three less pathogenic E. coli isolates. At 6 h postinoculation, all isolates had colonized the respiratory tract (trachea, lungs, and air sacs) and internal organs (liver, spleen, and kidney) of inoculated birds, but bacteria were recovered from pericardial fluid and blood only of birds inoculated with the more pathogenic isolates. F1 fimbriae were expressed on a high proportion of bacteria colonizing the trachea and to a lesser extent on bacteria in the lungs of birds inoculated with each of the isolates. F1 fimbriae were also expressed on bacteria in air sacs only for the less pathogenic isolates. P(F11) fimbriae were expressed on bacteria present in air sacs, lungs, kidney, blood, and pericardial fluid of birds inoculated with one of the more virulent isolates. On electron microscopy, bacteria of the more pathogenic isolates but not of the less pathogenic isolates were observed often associated with or within macrophages, which appeared to be viable, in the air sacs and lungs. In in vitro assays, the more pathogenic but not the less pathogenic isolates, were resistant to opsonization and phagocytosis in the absence of F1 fimbriae, whereas bacteria of all isolates were rapidly killed by avian macrophages when they expressed F1 fimbriae. These results suggest that resistance to phagocytosis may be an important mechanism in avian colisepticemia. They also suggest that F1 fimbrial phase variation to the nonfimbriated phase is favored in the avian lower respiratory tract, is more marked for the more pathogenic-isolates, and may be a virulence mechanism.

Dynamics of Escherichia coli Infection in Experimentally Inoculated Chickens

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

Genotyping of Canadian isolates of fowl adenoviruses

Five hundred and seventy-three clinical submissions with fowl adenovirus (FAdV) involvement were examined to investigate the association of different types of FAdV with clinical problems related to FAdV infection. Samples were received from 2000 to 2006 and originated from seven Canadian provinces. Four hundred and eighty-seven submissions were inclusion body hepatitis (IBH) related, while 86 were not IBH related. Viruses isolated from 287 samples were further analysed by hexon gene loop 1 sequencing. Twenty-seven genotyped FAdVs were from Alberta, 20 from British Columbia, 16 from Manitoba, one from Nova Scotia, 82 from Ontario, 64 from Quebec and 77 from Saskatchewan. Two hundred and forty-six analysed FAdVs were from IBH cases, confirmed by liver histopathology, by FAdV isolation from the liver, or both. Based on hexon gene loop 1 sequencing analysis, FAdVs associated with IBH outbreaks were genetically related to FAdV02 (nine isolates, 99.4%), FAdV08a (100 isolates, 99.4% to 100%) and FAdV11 (98 isolates, 99.4% to 100%). Thirty-nine viruses were 93.7% to 94.3% identical to FAdV07 strain x11a, but the genetic and immunogenic properties of this strain require further investigation. In IBH cases, the co-infection rates for infectious bursal disease virus, infectious bronchitis virus, reoviruses and Newcastle disease virus were 3.47%, 1.04%, 6.25% and 0.69%, respectively. Forty-one genotyped FAdVs were from “non-IBH” cases. Viruses isolated from non-IBH cases consisted of 22 FAdV01, 15 FAdV11, two FAdV08a and one each of FAdV02 and FAdV04 viruses. Co-infection rates in non-IBH submissions were 50.00% for infectious bursal disease virus, 40.70% for infectious bronchitis virus, 27.91% for reoviruses and 1.16% for Newcastle disease virus.

Prevalence and Risk Factors for Salmonella and Campylobacter spp. Carcass Contamination in Broiler Chickens Slaughtered in Quebec, Canada

An observational study was conducted to estimate prevalence and risk factors for Salmonella and Campylobacter spp. carcass contamination in broiler chickens. Eighty-two lots were sampled in four slaughterhouses located in the province of Québec, Canada, over a 10-month period. Carcass contamination was evaluated by the carcass rinse technique for about 30 birds per lot. Exposure to potential risk factors was evaluated based on data from questionnaires, meteorology, and cecal cultures. Multivariable binomial negative regression models were used for risk factor analysis at the lot level. The prevalence of Salmonella-positive carcasses was 21.2% (95% confidence interval: 15.7 to 26.7%). Significant risk factors (P < 0.05) associated with a higher proportion of positive carcasses within lots were Salmonella-positive cecal culture, low rainfall during transportation to the slaughterhouse, temperature of > or = 0 degree C during transportation to the slaughterhouse, and a > or = 4-h waiting period in shipping crates before slaughtering. The prevalence of Campylobacter-positive carcasses was 35.8% (95% confidence interval: 27.1 to 44.5%). Lots containing birds with Campylobacter-positive cecal culture results, lots of birds that were slaughtered at the end of the week, and lots with at least 20% of birds with digestive contents detected in the jejunum at time of slaughtering had a significantly higher proportion (P < 0.05) of contaminated carcasses. These results support the importance of preharvest control measures implemented during rearing to reduce contamination of the final product. Weather during transportation to slaughter and the day of the week that birds were slaughtered also were associated with carcass contamination; further studies are needed to determine the underlying mechanisms by which these factors influence carcass contamination.

Multiple-antibiotic resistance of Enterococcus faecalis and Enterococcus faecium from cecal contents in broiler chicken and turkey flocks slaughtered in Canada and plasmid colocalization of tetO and ermB genes.

This study was conducted to characterize the antimicrobial resistance determinants and investigate plasmid colocalization of tetracycline and macrolide genes in Enterococcus faecalis and Enterococcus faecium from broiler chicken and turkey flocks in Canada. A total of 387 E. faecalis and E. faecium isolates were recovered from poultry cecal contents from five processing plants. The percentages of resistant E. faecalis and E. faecium isolates, respectively, were 88.1 and 94% to bacitracin, 0 and 0.9% to chloramphenicol, 0.7 and 14.5% to ciprofloxacin, 72.6 and 80.3% to erythromycin, 3.7 and 41% to flavomycin, 9.6 and 4.3% (high-level resistance) to gentamicin, 25.2 and 17.1% (high-level resistance) to kanamycin, 100 and 94% to lincomycin, 0 and 0% to linezolid, 2.6 and 20.5% to nitrofurantoin, 3 and 27.4% to penicillin, 98.5 and 89.7% to quinupristin-dalfopristin, 7 and 12.8% to salinomycin, 46.7 and 38.5% (high-level resistance) to streptomycin, 95.6 and 89.7% to tetracycline, 73 and 75.2% to tylosin, and 0 and 0% to vancomycin. One predominant multidrug-resistant phenotypic pattern was identified in both E. faecalis and E. faecium (bacitracin, erythromycin, lincomycin, quinupristin-dalfopristin, tetracycline, and tylosin). These isolates were further examined by PCR and sequencing for the genes encoding their antimicrobial resistance. Various combinations of vatD, vatE, bcrR, bcrA, bcrB, bcrD, ermB, msrC, linB, tetM, and tetO genes were detected, and ermB, tetM, and bcrB were the most common antimicrobial resistance genes identified. For the first time, plasmid extraction and hybridization revealed colocalization of tetO and ermB genes on a ca. 11-kb plasmid in E. faecalis isolates, and filter mating experiments demonstrated its transferability. Results indicate that the intestinal enterococci of healthy poultry, which can contaminate poultry meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes.

Impact of a drug-free program on broiler chicken growth performances, gut health, Clostridium perfringens and Campylobacter jejuni occurrences at the farm level

The use of antimicrobial agents as feed additives in poultry production is a public health concern due to the overall increase in antimicrobial resistance. Although some alternative products are commercially available, little is known on their potential impact on flock health and productivity. A prospective study involving 1.55 million birds was conducted on eight commercial broiler farms in Québec, Canada, to evaluate the impact of replacing antibiotic growth promoters and anticoccidial drugs by a drug-free program including improved brooding conditions, anticoccidial vaccination, essential oil-based feed additives, and water acidification. Various productivity and health parameters were compared between barns allocated to the conventional and the drug-free program. Zootechnical performances were monitored as productivity criteria. Clinical necrotic enteritis and subclinical enteritis occurrences, litter and fecal moistures content were measured, and microscopic gut health was evaluated. Clostridium perfringens and Campylobacter spp. strains were recovered from fecal samples collected during farm visits. Clostridium perfringens counts were used as poultry health indicators and Campylobacter prevalence was noted as well. The drug-free program was associated with a significant increase in feed conversion ratio and a decrease in mean live weight at slaughter and in daily weight gain. An increased incidence of necrotic enteritis outbreaks and subclinical enteritis cases, as well as an increase in litter moisture content at the end of the rearing period were also observed for this program. Mean microscopic intestinal lesion scores and prevalence of Campylobacter colonization were not statistically different between the two groups but the drug-free program was associated with higher Clostridium perfringens isolation rates. According to the current study design, the results suggest that substitution of antibiotic growth promoters and anticoccidial drugs by a drug-free program impacts various broiler chicken production parameters and Clostridium perfringens carriage levels.

Efficacy of Egg Cleaning Compounds on Eggshells Contaminated with Salmonella enterica Serovar Enteritidis

Salmonella Enteritidis infections of egg contents can be related to external contamination of the shell. In this study, the efficacy of three commercial cleaning and/or sanitizing compounds (sodium carbonate, sodium hypochlorite, and potassium hydroxide) was evaluated for bactericidal activity at pH values of 10, 11, and 12 against various concentrations (10(2), 10(4), or 10(6) CFU/ml) of Salmonella Enteritidis inoculated onto the eggshell surface. Efficacy of these chemical agents was also assessed against Salmonella Enteritidis in aqueous suspension. Our results indicated that none of the chemicals applied at the recommended manufacturers concentrations (sodium carbonate, 36 ppm; other treatments, 200 ppm) could eliminate Salmonella Enteritidis from eggshells artificially contaminated with the highest bacterial concentrations (10(4) or 10(6) CFU/ml). Higher concentrations of each product, at least 5 to 20 times greater than recommended doses, were needed to destroy the bacteria on egg surfaces. However, at or slightly above the manufacturers recommended use concentrations, all three formulations were effective against Salmonella Enteritidis in aqueous suspension (10(8) CFU/ml) or on eggshells contaminated with 10(2) CFU/ml. For both shell and suspension assays, inactivation of Salmonella Enteritidis occurred at lower concentrations at pH 12 than at pH 11 and 10. Contact time between chemicals and Salmonella apparently influenced bacterial inactivation. Extended contact times (2 to 10 min) reduced minimum chemical concentrations necessary to inactivate the bacteria. However, neither pH nor contact time influenced Salmonella Enteritidis inactivation when the initial bacterial numbers on eggshells were high.

Cellulitis in broiler chickens: a one-year retrospective study in four Québec abattoirs.

A 1-yr retrospective study was undertaken to verify whether the prevalence of cellulitis in broiler chicken flocks was associated 1) with sex and average body weight controlling for the time of year and 2) with some other condemnation causes such as air-sacculitis, ascites, cyanosis, emaciation, valgus varus deformity, peritonitis, and total condemnation rate controlling for sex, average body weight, and time of year. Data were collected from four Québec abattoirs between October 1992 and September 1993. Data from 2452 unisex male and female broiler flocks were compiled and analyzed with univariate and multivariate models. The prevalence of cellulitis among flocks was 42.2 per 10,000 birds slaughtered. Cellulitis was associated with sex and increased with time of year (P < 0.0001). The prevalence was higher in male (mean +/- SD, 50.7+/-24.3; median, 31.8) than in female flocks (mean +/- SD, 34.2+/-15.6; median, 20.0) but showed no relationship with average body weight. Associations between condemnation causes and cellulitis were relatively weak; increases in the prevalence of cellulitis correlated with increases in total condemnation rate (r2 = 0.19), ascites (r2 = 0.09), airsacculitis (r2 = 0.0), cyanosis (r2 = 0.04), peritonitis (r2 = 0.03), emaciation (r2 = 0.02), and valgus varus deformity (r2 = 0.02). These results suggest that flock sex is a potential correlate of cellulitis prevalence in broiler chickens. Associations between the prevalence of cellulitis and other diseases observed at the abattoir were not very strong. However, it appears here that general disease status of Québec broiler flocks is a better indicator of cellulitis occurrence than the main diseases considered individually.

Evaluation of the Adhesive Capacity of Escherichia coli Isolates Associated with Avian Cellulitis

SUMMARY. It has been shown that Escherichia coli isolates from lesions of cellulitis belong to a limited number of clonal groups distinct from those of isolates found in the environment of these birds. In this study, different in vitro methods were used to evaluate adherence properties of E. coli isolates from cellulitis lesions and environments of high- and low-cellulitis prevalence broiler flocks. One hundred isolates were tested by hemagglutination. Adherence to frozen sections of chicken skin and binding to soluble fibronectin were examined for 40 of these 100 isolates by immunofluorescence and by immunocytofluorometry, respectively. Localization of bacterial adherence to skin tissues was confirmed by immunohistochemistry. It was demonstrated that O78:K80 isolates from cellulitis lesions adhered to skin sections to a much greater extent in deeper than in superficial tissue layers. A greater bacterial adherence following growth in TSB at 37 C was demonstrated for isolates from flocks with high prevalence of cellulitis than for isolates from flocks with low prevalence of cellulitis. MANOVA analysis results showed a significant difference between superficial and deep tissue layers only for one set of isolates from flocks with high prevalence of cellulitis. Hemagglutinating activity was variable among the O78:K80 isolates obtained from flocks with high prevalence of cellulitis. The results obtained for some O78:K80 isolates following growth in TSB suggest a role for type 1 fimbriae or F1 in adherence to skin sections. This was reinforced by the finding that adherence was inhibited by d-mannose. Poultry E. coli isolates that express F1 had no affinity for soluble fibronectin, although localization of the adherence in skin sections suggested a role for extracellular matrix components such as collagen and insoluble fibronectin.