Post-weaning diarrhea is a major health challenge in the swine industry and is routinely managed by fortifying pig starter diets with antimicrobials. But there are concerns about antibiotic resistance, hence the need for identifying effective alternatives. The use of spray-dried whole egg powder containing anti-F4 antibodies (SDWE) against recombinant F4 antigens and chitosan oligosaccharide and Enterococcus fecalis probiotic combination (CPRO) was investigated in two trials using enterotoxigenic Escherichia coli K88+ (ETEC) oral challenge model in 21-d-old piglets. Pre-challenge, SDWE supported higher (P < 0.05) piglet performance whereas during the post-challenge period, SDWE and CPRO had no effect on growth performance but diarrhea incidences and severity were reduced (P > 0.05) in SDWE-fed piglets compared to the control. The results show that SDWE supported greater piglet performance pre-ETEC challenge although there was no benefit of SDWE or CPRO supplementation evident during the post-challenge period in early-weaned pigs. / October 2015

Entrotoxigenic Escherichia coli (ETEC) K88 is a causative agent of post-weaning diarrhea (PWD) in early-weaned pigs. This study investigated the efficacy of two alternative diets, charcoal (0.1, 0.5, 1, and 2%) and a low crude protein (CP) diet (17%) supplemented with probiotic E. coli strains (UM2 and UM7), against PWD infection in ETEC K88 challenged piglets. The present study found that charcoal had no effect on the challenged piglets’ performance, ileal and colonic microbiota or their fermentation end products. There was, however, a correlation between charcoal dosage and fecal consistency score. Charcoal reduced the ileal mucosal attached ETEC K88. Feeding a low-CP diet resulted in a lower ileal ammonia concentration. The low-CP diet reduced the E. coli populations in the ileal digesta as well as lowered mRNA expression of the IL-1ß. We concluded that the use of both 1-2% charcoal diet and a low-CP diet supplemented with probiotic E. coli strains were effective in reducing the incidence and severity of PWD infection.

Entrotoxigenic Escherichia coli (ETEC) K88 is a causative agent of post-weaning diarrhea (PWD) in early-weaned pigs. This study investigated the efficacy of two alternative diets, charcoal (0.1, 0.5, 1, and 2%) and a low crude protein (CP) diet (17%) supplemented with probiotic E. coli strains (UM2 and UM7), against PWD infection in ETEC K88 challenged piglets. The present study found that charcoal had no effect on the challenged piglets’ performance, ileal and colonic microbiota or their fermentation end products. There was, however, a correlation between charcoal dosage and fecal consistency score. Charcoal reduced the ileal mucosal attached ETEC K88. Feeding a low-CP diet resulted in a lower ileal ammonia concentration. The low-CP diet reduced the E. coli populations in the ileal digesta as well as lowered mRNA expression of the IL-1ß. We concluded that the use of both 1-2% charcoal diet and a low-CP diet supplemented with probiotic E. coli strains were effective in reducing the incidence and severity of PWD infection.

No description available.

BACKGROUND: Vibrio cholerae and Enterotoxigenic E. Coli (ETEC) are enteropathogens that are global causes of cholera and traveler’s diarrhea which are responsible for millions of diarrhea cases every year. ETEC and cholera are primarily found in Sub-Saharan Africa and Asia, particularly in nations with inadequate sanitation systems or little access to clean water. Infants and children are most vulnerable to these diseases, as severe infections can lead to stunting and death. The incidence of cholera and ETEC diarrhea have increased, due in part to changing weather patterns. At present, robust animal models for studies of ETEC colonization are lacking to study colonization and bottlenecks. The only licensed vaccines against cholera in endemic countries are killed whole cells, however, new live attenuated oral cholera vaccines (OCV) are in development and offer significant advantages. PanChol is a live attenuated OCV entering phase I trials. SPECIFIC AIMS: To propel studies of ETEC pathogenesis, I attempted to create a suckling mouse model of this globally important pathogen. To accomplish this goal, I constructed barcoded ETEC libraries that enabled me to determine founding population sizes along with intestinal ETEC burdens. To better understand PanChol, a new live attenuated OCV, I studied the shedding of the vaccine in the first 3 human volunteers to ingest this novel agent. METHODS: Triparental mating of donor strains MFDλpir pJMP1039 and MFDλpir pSM1 with recipient ETEC strains enabled construction of barcoded libraries. Neonatal CD-1 and C57BL/6 mice were infected with 104-107 CFU of wild-type ETEC to develop an infant mouse model. Founding population sizes of ETEC strains were compared via sequencing and STAMPR analysis while CFU burdens were determined via plating. Shedding of PanChol was done through enumeration of serial dilutions of fecal samples. Serotyping of shed PanChol was carried out using anti-Ogawa and anti-Inaba antisera. RESULTS: There were marked differences in ETEC small intestinal colonization in different mouse strains. Outbred CD-1 suckling mice only colonized with a 107 dose. In contrast, colonization of ETEC was approximately 106 CFU/small intestine at inocula sizes of 105 or greater in C57BL/6 mice. Laboratory studies using simulated bottlenecks made by serial dilutions established that the barcoded libraries accurately reflect founding population sizes up to 105 CFU. There was no difference in founding population sizes at the same inoculum size between WT ETEC and a hypervesiculation ∆mlaE mutant, though the founding population size increased with increasing input. PanChol retained the Hikojima serotype and shedding occurred in all volunteers with maximum colonization occurring 3 days post administration of 106 CFU. CONCLUSIONS: C57BL/6 P5 mice can serve as a new model to study ETEC intestinal colonization. Hypervesiculating ETEC did not produce a difference in founding population or colonization at the same input as WT ETEC strains. PanChol shows great promise as a viable OCV with shedding at 106 input and no serotype reversion.

Linhagens de Escherichia coli enterotoxigênica (ETEC) são consideradas como importante agente de diarréia, principalmente entre crianças e turistas em países em desenvolvimento. Entre os fatores de virulência expressos por ETEC, as enterotoxinas termo-lábil (LT) e termo-estável (ST) representam os mais relevantes fenótipos. Evidências preliminares sugerem que a severidade da diarréia associada a linhagens de ETEC deve refletir a diversidade natural de linhagens selvagens quanto à produção de enterotoxinas e/ou à ocorrência de variantes naturais com efeitos tóxicos reduzidos. No presente trabalho, investigamos diversidade genética do óperon etx, que codifica para a toxina LT, e da capacidade de produção e secreção de LT por linhagens de ETEC isoladas de humanos ou suínos em diferentes regiões geográficas. Os resultados mostraram considerável variabilidade na produção de LT com valores variando de 2 a 2.525 ng de toxina por mL de cultura. Secreção de LT foi também variável com valores variando de menos que 0,04% a 49,5% do total de LT produzida pelas diferentes linhagens de ETEC. Adicionalmente, experimentos de alça ligada em coelho mostraram uma boa correlação entre a quantidade de LT secretada sob condições in vitro e a capacidade de causar acúmulo de fluidos in vivo. Nós determinamos ainda diversidade de ETEC pela obtenção das seqüências dos óperons etxAB de 50 linhagens (LT+ or LT+/ST+) pertencentes a diferentes sorotipos com ênfase para as linhagens produtoras apenas de LT e isoladas de crianças assintomáticas. As seqüências de nucleotídeo completas dos genes etxAB revelaram 23 alterações de aminoácidos nas subunidades A (18) e B (5), as quais geraram 16 variantes de LT. Entre estes variantes de LT, um mostrou efeito tóxico reduzido em comparação à toxina de referência LT1. A forma de LT atenuada (LT4) tem atividade enzimática reduzida devido à troca de aminoácido. / Enterotoxigenic Escherichia coli (ETEC) strains represent an important etiological agent of diarrheal disease, particularly among children and travelers in developing countries. Among the virulence factors expressed by ETEC strains the heat-labile (LT) and heat-stable (ST) enterotoxins represent the most revelevant phenotypes. Indirect evidences suggest that the severity of diarrhea associated to ETEC strains might reflect the natural diversity of wild strains to produce enterotoxins and/or the occurrence of variants endowed with reduced toxic effects. In the present study, we investigated both the genetic diversity of the etx operon, encoding the heat-labile toxin, and the capability to produce/secrete LT by ETEC strains isolated from humans or porcine in different geoghrafic areas. The results showed a remarkable variability on the production of LT with values ranging from 2 to 2,525 ng of toxin per ml of culture. LT secretion was also variable with values ranging from less than 0.04% to 49.5% of total LT produced by the different ETEC strains. Additionally, rabbit ileal loop experiments showed a good correlation between the amounts of secreted LT under in vitro conditions and fluid accumulation in vivo. We determined also the diversity of the etxAB operon of 50 ETEC strains (LT+ or LT+/ST+) belonging to different serotypes with emphasis to LT+-only producing strains isolated from asymptomatic children. The complete nucleotide sequences of the etxAB genes revealed 23 amino acid changes at the A (18) or B (5) subunits, which generated 16 variant forms of LT. Among these LT variants, one of them showed reduced toxic effects in comparison to the reference toxin LT1. The attenuated LT form (LT4) had decreased enzymatic activity due to an amino acid replacement (K4R) at the A1 subunit. LT4 retains its immunogenic and adjuvant properties following nasal immunization. Additionaly, the LT4 variant showed altered immune modulatory features and promoted a more biased Th1 response, which favor activation of effector CD8+ T lymphocytes, to co-administred antigen with regard to LT1. Taken together, our results demonstrate that ETEC strains isolated from human subjects express natural genetic variability leading to a remarkable polymorphism of the etx operon as well as production and secretion of LT. Such natural genetic diversity observed in ETEC strains may affect the host-pathogen relationships and, consequently, contribute to the severity of the disease among infected subjects.

Linhagens de Escherichia coli enterotoxigênica (ETEC) são consideradas como importante agente de diarréia, principalmente entre crianças e turistas em países em desenvolvimento. Entre os fatores de virulência expressos por ETEC, as enterotoxinas termo-lábil (LT) e termo-estável (ST) representam os mais relevantes fenótipos. Evidências preliminares sugerem que a severidade da diarréia associada a linhagens de ETEC deve refletir a diversidade natural de linhagens selvagens quanto à produção de enterotoxinas e/ou à ocorrência de variantes naturais com efeitos tóxicos reduzidos. No presente trabalho, investigamos diversidade genética do óperon etx, que codifica para a toxina LT, e da capacidade de produção e secreção de LT por linhagens de ETEC isoladas de humanos ou suínos em diferentes regiões geográficas. Os resultados mostraram considerável variabilidade na produção de LT com valores variando de 2 a 2.525 ng de toxina por mL de cultura. Secreção de LT foi também variável com valores variando de menos que 0,04% a 49,5% do total de LT produzida pelas diferentes linhagens de ETEC. Adicionalmente, experimentos de alça ligada em coelho mostraram uma boa correlação entre a quantidade de LT secretada sob condições in vitro e a capacidade de causar acúmulo de fluidos in vivo. Nós determinamos ainda diversidade de ETEC pela obtenção das seqüências dos óperons etxAB de 50 linhagens (LT+ or LT+/ST+) pertencentes a diferentes sorotipos com ênfase para as linhagens produtoras apenas de LT e isoladas de crianças assintomáticas. As seqüências de nucleotídeo completas dos genes etxAB revelaram 23 alterações de aminoácidos nas subunidades A (18) e B (5), as quais geraram 16 variantes de LT. Entre estes variantes de LT, um mostrou efeito tóxico reduzido em comparação à toxina de referência LT1. A forma de LT atenuada (LT4) tem atividade enzimática reduzida devido à troca de aminoácido. / Enterotoxigenic Escherichia coli (ETEC) strains represent an important etiological agent of diarrheal disease, particularly among children and travelers in developing countries. Among the virulence factors expressed by ETEC strains the heat-labile (LT) and heat-stable (ST) enterotoxins represent the most revelevant phenotypes. Indirect evidences suggest that the severity of diarrhea associated to ETEC strains might reflect the natural diversity of wild strains to produce enterotoxins and/or the occurrence of variants endowed with reduced toxic effects. In the present study, we investigated both the genetic diversity of the etx operon, encoding the heat-labile toxin, and the capability to produce/secrete LT by ETEC strains isolated from humans or porcine in different geoghrafic areas. The results showed a remarkable variability on the production of LT with values ranging from 2 to 2,525 ng of toxin per ml of culture. LT secretion was also variable with values ranging from less than 0.04% to 49.5% of total LT produced by the different ETEC strains. Additionally, rabbit ileal loop experiments showed a good correlation between the amounts of secreted LT under in vitro conditions and fluid accumulation in vivo. We determined also the diversity of the etxAB operon of 50 ETEC strains (LT+ or LT+/ST+) belonging to different serotypes with emphasis to LT+-only producing strains isolated from asymptomatic children. The complete nucleotide sequences of the etxAB genes revealed 23 amino acid changes at the A (18) or B (5) subunits, which generated 16 variant forms of LT. Among these LT variants, one of them showed reduced toxic effects in comparison to the reference toxin LT1. The attenuated LT form (LT4) had decreased enzymatic activity due to an amino acid replacement (K4R) at the A1 subunit. LT4 retains its immunogenic and adjuvant properties following nasal immunization. Additionaly, the LT4 variant showed altered immune modulatory features and promoted a more biased Th1 response, which favor activation of effector CD8+ T lymphocytes, to co-administred antigen with regard to LT1. Taken together, our results demonstrate that ETEC strains isolated from human subjects express natural genetic variability leading to a remarkable polymorphism of the etx operon as well as production and secretion of LT. Such natural genetic diversity observed in ETEC strains may affect the host-pathogen relationships and, consequently, contribute to the severity of the disease among infected subjects.

La résistance aux antibiotiques a pris une ampleur considérable du fait de l’utilisation des antibiotiques dans de nombreux domaines. Pour respecter l’approche « OneHealth », il est essentiel d’avoir une image spécifique de chaque situation, afin d’orienter les recommandations et de limiter la dissémination des gènes, des plasmides et des clones. Nos objectifs étaient adaptés à nos populations d’étude (chevaux et porcs) afin d’ajuster les résultats aux besoins des filières. Dans la filière équine, nous avons quantifié les résistances phénotypiques et identifié les gènes de β-lactamases à spectre étendu (BLSE/AmpC) présents dans le microbiome des chevaux sains, et nous avons identifié les facteurs de risque associés à leur portage en France et au Québec. En France, nous avons également caractérisé les mécanismes de dissémination des gènes de BLSE/AmpC. Nous avons mis en évidence qu’en France et au Québec, les E. coli commensaux provenant de fèces de chevaux sains étaient majoritairement non susceptibles à l’ampicilline, l’amoxicilline/acide clavulanique et la streptomycine et que des E. coli multirésistants et porteurs de gènes codant pour des BLSE/AmpC étaient détectés dans respectivement environ 45% et 8% des chevaux. Le blaCTX-M-1 était majoritairement détecté bien qu’en France d’autres BLSE aient été identifiés (blaCTX-M-2 et blaCTX-M-14) ainsi que le gène AmpC blaCMY-2. L’administration d’un traitement médical, le nombre de personnes s’occupant des chevaux, le type d’activité et le fait de participer à un évènement équestre dans les trois derniers mois ont été identifiés comme des facteurs de risque du portage des E. coli multirésistants ou producteurs de gènes BLSE/AmpC, soit en France soit au Québec. En France, le plasmide IncHI1-ST9 était majoritairement associé aux gènes blaCTX-M-1/2 et à l’opéron fos. Pour la filière porcine, nos objectifs étaient de colliger les données de la base de données du laboratoire EcL entre 2008 et 2016, d’évaluer la présence d’un agrégat spatio-temporel pour les isolats ETEC:F4 non susceptibles à l’enrofloxacine et de caractériser ces isolats et les éléments génétiques mobiles qu’ils transportent. En effet, l’enrofloxacine est un antibiotique de haute importance en santé humaine, et doit donc faire l’objet d’une surveillance accrue. Nous avons trouvé que plus de 90% des isolats d’E. coli entérotoxinogènes détectés chez des cas de porcs malades soumis au laboratoire EcL de 2008 à 2016 au Québec, étaient multirésistants. Le virotype LT:STb:F4 prédominait jusqu’en 2014, puis a été dépassé par le virotype LT:STb:STa:F4. Un agrégat spatio-temporel d’isolats LT:STb:STa:F4 non susceptibles à l’enrofloxacine a été détecté entre 04/2015 et 09/2016 au centre de la Montérégie. Nous avons démontré la présence d’un clone ETEC:F4 non susceptible à l’enrofloxacine, à haut risque, qui se dissémine en Amérique du Nord depuis 2013. Les isolats appartenant à ce clone sont ST100, O149:H10. Ils sont multirésistants, et associés à une pathogénicité et une virulence augmentée par rapport aux isolats détectés avant 2000. Ils portent le réplicon IncFII. Les résistances et leur mécanisme de dissémination sont différents selon l’espèce animale. Ces divergences sont fonction de l’usage des antibiotiques, et des échanges possibles avec les différents protagonistes en contact avec les animaux. / Antimicrobial resistance has become an essential issue in the last decades because of the extensive use of antimicrobials in numerous sectors. In order to follow the OneHealth approach, it is critical to have a precise picture of each situation, to adjust recommendations and prevent resistance gene dissemination as well as plasmid and clone spread. Our objectives were adapted to the animal populations under study. Therefore, our results were compatible with each sector. In the equine sector, we quantified phenotypic resistance and identified β-lactamase (ESBL/AmpC) genes present in the intestinal microbiome of healthy horses and we identified risk factors associated with their carriage both in France and in Quebec. Then, in France we characterized ESBL/AmpC gene spread mechanisms. We demonstrated that commensal E. coli originating from the feces of healthy horses were mostly non-susceptible to ampicillin, amoxicillin/clavulanic acid and streptomycin. The presence of multidrug resistant E. coli and of E. coli carrying ESBL/AmpC genes was found in around 45% and 8% of horses respectively. The most frequently detected gene was blaCTX-M-1, although blaCTX-M-2 and blaCTX-M-14 were also identified in France. The AmpC gene blaCMY-2 was identified in both localities. Medical treatment, staff number, activity, and participation in an equestrian event within the last three months were identified as risk factors for MDR or ESBL/AmpC E. coli. In France, commensal E. coli from healthy horses most commonly possessed the IncHI1-ST9 plasmid. This plasmid carries blaCTX-M-1/2 genes and the fos operon. For the swine sector in Quebec, our objectives were to gather data provided by the Animal pathogenic and zoonotic E. coli (APZEC) database between 2008 and 2017, to assess the presence of a spatio-temporal cluster for enrofloxacin non-susceptible ETEC:F4 and to characterize these isolates and the mobile genetic elements they carry. Enrofloxacin is an antibiotic classified as highly important in human medicine and as such needs to come under higher scrutiny. For this sector, we demonstrated that more than 90% of enterotoxigenic E. coli (ETEC) isolates from diseased swine submitted to the EcL between 2008 and 2016, were multidrug resistant. The main virotype in 2014 was LT:STb:F4. It was subsequently replaced by the LT:STb:STa:F4 virotype. A spatio-temporal cluster of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin was detected between 04/2015 and 09/2016 in the centre of the Monteregie region. These isolates constituted an ETEC:F4 high risk enrofloxacin non-susceptible clone, which has been spreading in North America since 2013. Isolates belonging to this clone are ST100, O149H10, phylogroup A, and fimH gene negative. These isolates are multidrug resistant and associated with a higher pathogenicity and virulence than isolates detected before 2000. They all carry the incFII replicon. Resistance and mechanisms of dissemination are different according to the animal species being studied. This is likely due to different patterns of antimicrobial use in each industry and possible interactions with different protagonists in contact with the animals. It is essential to understand the situation for each animal species in order to adapt recommendations for efficiently limiting the spread of resistance genes, plasmids and clones.

A major challenge for the pig industry is to formulate starter diets that primarily fit the digestive capacity, maintain GIT health and promote growth without recourse to in-feed antimicrobials. Experiments were conducted to evaluate the efficacy of carbohydrase enzymes (CE) targeting non-starch polysaccharides (NSP) in enhancing gut health and function in piglets. First, an experiment was conducted to evaluate the effects of adding CE in piglet diets on growth performance, GIT bacterial activity and nutrient digestibility. Pigs fed diets containing CE had a higher ileal lactobacilli count, total organic acids concentrations, NSP digestibility and low ammonia compared with control. The effectiveness of CE targeting NSP was further evaluated using enterotoxigenic E. coli (ETEC) in a challenge model to evaluate the impact on gut health and function. Two approaches for the ETEC challenge were adopted; an in situ small intestine segments perfusion model and an in vivo model. Initially, a pilot study was conducted to establish and validate the in situ model. In the pilot study, conventional anti-diarrhea agents; fumaric acid, ZnO, egg yolk antibodies against ETEC K88 fimbriae and carbadox, attenuated fluid losses in ETEC-infected jejunal segments. Following the establishment of the in situ model, four experiments were conducted to study the effects NSP hydrolysis products (HP) from various feedstuffs (i.e. wheat, soybean meal, canola meal and flaxseed) on ETEC-induced secretory diarrhea. The results demonstrated that HP protected against ETEC-induced fluid and electrolyte losses. A further study was conducted to investigate the response of piglets fed diets containing HP and EYA singly or in combination upon oral challenge with ETEC. Feeding HP and EYA alone or in combination attenuated ETEC-enteritis symptoms such that piglets fed additives showed less pronounced acute phase responses and superior performance. Piglets fed diets containing additives had lower gastric pH, fewer ETEC adhered to ileal mucosa and lower incidence of diarrhea. Overall, reduction of intestinal pathogens or toxic bacterial metabolites contributes to enhanced GIT health and function. These novel results expand the scope of enzyme technology in animal nutrition within the new paradigm of dietary approaches to gut health and function.

A major challenge for the pig industry is to formulate starter diets that primarily fit the digestive capacity, maintain GIT health and promote growth without recourse to in-feed antimicrobials. Experiments were conducted to evaluate the efficacy of carbohydrase enzymes (CE) targeting non-starch polysaccharides (NSP) in enhancing gut health and function in piglets. First, an experiment was conducted to evaluate the effects of adding CE in piglet diets on growth performance, GIT bacterial activity and nutrient digestibility. Pigs fed diets containing CE had a higher ileal lactobacilli count, total organic acids concentrations, NSP digestibility and low ammonia compared with control. The effectiveness of CE targeting NSP was further evaluated using enterotoxigenic E. coli (ETEC) in a challenge model to evaluate the impact on gut health and function. Two approaches for the ETEC challenge were adopted; an in situ small intestine segments perfusion model and an in vivo model. Initially, a pilot study was conducted to establish and validate the in situ model. In the pilot study, conventional anti-diarrhea agents; fumaric acid, ZnO, egg yolk antibodies against ETEC K88 fimbriae and carbadox, attenuated fluid losses in ETEC-infected jejunal segments. Following the establishment of the in situ model, four experiments were conducted to study the effects NSP hydrolysis products (HP) from various feedstuffs (i.e. wheat, soybean meal, canola meal and flaxseed) on ETEC-induced secretory diarrhea. The results demonstrated that HP protected against ETEC-induced fluid and electrolyte losses. A further study was conducted to investigate the response of piglets fed diets containing HP and EYA singly or in combination upon oral challenge with ETEC. Feeding HP and EYA alone or in combination attenuated ETEC-enteritis symptoms such that piglets fed additives showed less pronounced acute phase responses and superior performance. Piglets fed diets containing additives had lower gastric pH, fewer ETEC adhered to ileal mucosa and lower incidence of diarrhea. Overall, reduction of intestinal pathogens or toxic bacterial metabolites contributes to enhanced GIT health and function. These novel results expand the scope of enzyme technology in animal nutrition within the new paradigm of dietary approaches to gut health and function.