Éric Nadeau

Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies.

Abstract Escherichia coli is one of the most important causes of postweaning diarrhea in pigs. This diarrhea is responsible for economic losses due to mortality, morbidity, decreased growth rate, and cost of medication. The E. coli causing postweaning diarrhea mostly carry the F4 (K88) or the F18 adhesin. Recently, an increase in incidence of outbreaks of severe E. coli-associated diarrhea has been observed worldwide. The factors contributing to the increased number of outbreaks of this more severe form of E. coli-associated diarrhea are not yet fully understood. These could include the emergence of more virulent E. coli clones, such as the O149:LT:STa:STb:EAST1:F4ac, or recent changes in the management of pigs. Development of multiple bacterial resistance to a wide range of commonly used antibiotics and a recent increase in the prevalence and severity of the postweaning syndromes will necessitate the use of alternative measures for their control. New vaccination strategies include the oral immunization of piglets with live avirulent E. coli strains carrying the fimbrial adhesins or oral administration of purified F4 (K88) fimbriae. Other approaches to control this disease include supplementation of the feed with egg yolk antibodies from chickens immunized with F4 or F18 adhesins, breeding of F18- and F4-resistant animals, supplementation with zinc and/or spray-dried plasma, dietary acidification, phage therapy, or the use of probiotics. To date, not a single strategy has proved to be totally effective and it is probable that the most successful approach on a particular farm will involve a combination of diet modification and other preventive measures.

Prevalence and comparison of genetic profiles of Campylobacter strains isolated from poultry and sporadic cases of campylobacteriosis in humans.

Between July 1998 and June 1999, 93 lots of broiler chickens distributed on 57 farms were sampled in two abattoirs of the province of Quebec (Canada). A total of 2,325 samples of cecal material were analyzed to determine the prevalence of campylobacters. Biotyping and pulsed-field gel electrophoresis (PFGE) were done on 20% of the Campylobacter isolates to study the distribution within poultry production. Macrorestriction profiles were compared with profiles of 24 Campylobacter strains isolated from sporadic cases of human diarrheic patients in order to evaluate genetic relationships. Approximately 40% of the broiler chickens in 60% of the lots and 67% of the farms were colonized. Biotypes I and II of Campylobacter jejuni were the most prevalent biotypes in poultry and human isolates. The PFGE dendograms revealed a high genetic diversity among poultry isolates, with 49 different genotypes from the 56 positive lots. More than 75% of these lots were colonized by a unique genotype. All positive lots raised simultaneously on the same farm had common genotype(s). Different genotypes were isolated from lots raised at different grow-out periods on a farm. In some cases, identical genotypes were found at different grow-out periods on a farm and also from different farms. Macrorestriction profiles showed that approximately 20% of human Campylobacter isolates were genetically related to genotypes found in poultry. This genetic relationship and the high prevalence of C. jejuni biotypes I and II in poultry indicated that Campylobacter in broiler production of the province of Quebec could be a potential source of hazard for public health.

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.

Administration of probiotics influences F4 (K88)-positive enterotoxigenic Escherichia coli attachment and intestinal cytokine expression in weaned pigs

This study evaluated the effect of the probiotics Pediococcus acidilactici and Saccharomyces cerevisiae boulardii on the intestinal colonization of O149 enterotoxigenic Escherichia coli harbouring the F4 (K88) fimbriae (ETEC F4) and on the expression of ileal cytokines in weaned pigs. At birth, different litters of pigs were randomly assigned to one of the following treatments: 1) control without antibiotics or probiotics (CTRL); 2) reference group in which chlortetracycline and tiamulin were added to weanling feed (ATB); 3) P. acidilactici; 4) S. cerevisiae boulardii; or 5) P. acidilactici + S. cerevisiae boulardii. Probiotics were administered daily (1 × 109 CFU per pig) during the lactation period and after weaning (day 21). At 28 days of age, all pigs were orally challenged with an ETEC F4 strain, and a necropsy was performed 24 h later. Intestinal segments were collected to evaluate bacterial colonization in the small intestine and ileal cytokine expressions. Attachment of ETEC F4 to the intestinal mucosa was significantly reduced in pigs treated with P. acidilactici or S. cerevisiae boulardii in comparison with the ATB group (P = 0.01 and P = 0.03, respectively). In addition, proinflammatory cytokines, such as IL-6, were upregulated in ETEC F4 challenged pigs treated with P. acidilactici alone or in combination with S. cerevisiae boulardii compared with the CTRL group. In conclusion, the administration of P. acidilactici or S. cerevisiae boulardii was effective in reducing ETEC F4 attachment to the ileal mucosa, whereas the presence of P. acidilactici was required to modulate the expression of intestinal inflammatory cytokines in pigs challenged with ETEC F4.

Comparison of Campylobacter Isolates from Poultry and Humans: Association between In Vitro Virulence Properties, Biotypes, and Pulsed-Field Gel Electrophoresis Clusters

ABSTRACT The in vitro virulence properties of 197 temporally and geographically related Campylobacter isolates from chicken broilers and humans were compared. Comparisons of the virulence properties associated with genotypes and biotypes were made. All isolates adhered to, and 63% invaded, INT-407 cells, whereas 13% were cytotoxic for CHO cells. CHO cell-cytotoxic extracts were also cytotoxic for INT-407 cells, but the sensitivity for Vero cells was variable. The proportion of isolates demonstrating a high invasiveness potential (>1,000 CFU ml−1) or Vero cell cytotoxicity was significantly higher for human than for poultry isolates. Invasiveness was associated with Campylobacter jejuni isolates of biotypes 1 and 2, whereas CHO and INT-407 cell cytotoxicity was associated with C. jejuni isolates of biotypes 3 and 4. Cytotoxic isolates were also clustered according to pulsed-field gel electrophoresis profiles.

Characterization of persistent and transient Escherichia coli isolates recovered from clinical mastitis episodes in dairy cows

Escherichia coli usually cause transient intramammary infections in dairy cows, but persistent intramammary infections have been observed. The objective of the study was to compare antimicrobial resistance and virulence genes found in persistent and transient E. coli isolated from clinical mastitis cases in a cohort of 91 Canadian dairy herds monitored over a 2-year period. Antimicrobial susceptibility was determined by broth microdilution and the presence of 27 virulence genes associated with extra-intestinal E. coli infections was determined by colony hybridization. Proportion of resistance in persistent E. coli ranged from 0.0% (enrofloxacin) to 27.8% (ampicillin and tetracycline). Proportion of resistance in transient E. coli ranged from 0.0% (enrofloxacin) to 16.8% (tetracycline). Odds of being classified as a persistent isolate increased by a factor of 1.6 (95% CI: 1.1, 2.4) for each aditional resistance observed (e.g. isolates resistant to four antimicrobial agents had 1.6 times higher odds of belonging to the persistent groups compared to isolates demonstrating resistance to three agents). Persistency was associated with higher odds of resistance to ampicillin (OR: 9.8, P<0.01) or cephalothin (OR: 7.6, P=0.02). Persistent isolates had 5.4 times higher odds (95% CI: 1.2, 24.0) of harboring virulence gene iroN. Similarly, persistent isolates had 8.6 times higher odds (95% CI: 2.8, 27.1) of possessing the virulence gene sitA. In conclusion, this study confirmed that persistency of intramammary E. coli isolates is associated with certain traits. Findings concerning iron-acquisition shed new light on the mechanisms of intramammary survival.

Oral Immunization with F4 Fimbriae and CpG Formulated with Carboxymethyl Starch Enhances F4-Specific Mucosal Immune Response and Modulates Th1 and Th2 Cytokines in Weaned Pigs

PURPOSE F4 fimbriae are a potential candidate for an oral subunit vaccine for prevention of post-weaning diarrhea in swine due to infection with F4-positive enterotoxigenic Escherichia coli. However, large quantities of F4 fimbriae are required to induce a specific antibody response. The aim of the present study was to evaluate the effect of supplementation of F4 fimbriae with Cytosine-phosphate-Guanosine-oligodeoxynucleotide (CpG-A D19) or with complete cholera toxin (CT) as adjuvants on the F4-specific antibody response and cytokine production in weaned pigs following oral administration of F4 fimbrial antigen formulated with Carboxymethyl Starch (CMS). METHODS Oral dosage forms of F4 fimbriae alone or supplemented with CpG-A D19 or with CT were formulated with CMS as monolithic tablets, obtained by direct compression, and administered to weaned pigs. Blood and faecal samples were collected to determine the systemic and mucosal immune status of animals at various times until necropsy. During necropsy, contents of the jejunum and ileum were collected for determination of mucosal F4 specific antibodies. Segments of jejunum and ileum were also used to measure mRNA cytokine production. RESULTS The presence of CpG in the formulation of the fimbriae significantly increased F4-specific immunoglobulin (Ig) IgM and IgG levels in intestinal secretions, and enhanced Th1 (Interferon-gamma / IFN-γ, Tumour Necrosis Factor-alpha / TNF-α, Interleukin-12p40 / IL-12p40, IL-1β) and Th2 (IL-4, IL-6) cytokine production in intestinal tissues. Supplementation with CT did not result in induction of F4-specific antibodies in secretions, although a significant Th1 response (IFN-α, IFN-γ, IL-18) was detected in tissues. Neither F4-specific systemic antibodies, nor intestinally secreted IgA were detected throughout the immunization trial for all groups. CONCLUSIONS CpG-A D19 appeared to be a promising adjuvant for an oral F4 subunit vaccine formulated with CMS excipient as monolithic tablets. This matrix afforded gastro-protection and delivered the F4 fimbriae at their intestinal sites.

Intestinal Mucosa Adherence and Cytotoxicity of a Sorbitol-Fermenting, Shiga-Toxin-Negative Escherichia coli O157:NM Isolate with an Atypical Type III Secretion System

Reports show that sorbitol-fermenting (SF) Escherichia coli O157 isolates are implicated in animal and human diseases and may represent new emerging pathogens. We investigated the cytotoxicity and interaction with intestinal tissues of an SF, Shiga-toxin-negative E. coli O157:NM isolate. This bovine isolate was negative for stx genes and was not cytotoxic for Vero cells. We found that this E. coli O157 isolate possesses an intimin of type beta, whereas the translocated intimin receptor Tir and type III secretion system components EspA, EspB, and EspD were of type alpha. In contrast, Shiga-toxin-positive O157:H7 isolates usually possess variants of type gamma. The isolate did not present typical O157:H7 attaching and effacing lesions in the newborn pig ileal in vitro organ culture model. However, extensive effacement and elongation of the microvilli were observed. In vitro organ culture results suggest that such an SF, Shiga-toxin-negative O157:NM isolate found in cattle may potentially cause disease, such as diarrhea without hemolytic uremic syndrome, in humans.