A rapid, sensitive method for testing the activity of <i>Escherichia coli</i> 0157:H7 secreted cytotoxins against epithelial cells from the jejunum and descending colon of cattle

Baines, Danica; Masson, Luke; McAllister, Tim A.

doi:10.4141/cjas07040

Cited by 6 publications

(10 citation statements)

References 19 publications

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“…The method for collecting the cytotoxins was previously described (Baines et al 2008). Briefly, the EHEC O157:H7 strains were grown in 15 mL of M9 media (Sigma-Aldrich, Oakville, ON) for 3 d. The culture was centrifuged for 30 min at 9299 )g, the supernatant collected and the pellet discarded.…”

Section: Ehec O157:h7 Secreted Cytotoxinsmentioning

confidence: 99%

“…The organ culture adherence assay was described previously (Baines et al 2008, manuscript in preparation). Generally, each 2.5-cm 2 organ piece was drained using a paper towel and placed in about 3 mL 158C DMEM (high glucose) adjusted to just cover the surface of the mucosa.…”

Section: Ivoc Adherence Assaymentioning

confidence: 99%

“…Key words: Escherichia coli O157:H7, cattle, intestine, cytotoxins, colonization Baines, D., Masson, L. et McAllister, T. 2008. Les cytotoxines se´cre´te´es par Escherichia coli O157:H7 sont toxiques pour les ente´rocytes et favorisent la colonisation du je´junum et du coˆlon descendant des bovins par le microorganisme.…”

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“…Shiga toxins are toxic to lymphocytes (Ferens and Hovde 2000) and heart endothelial cells (ValdiviesoGarcia et al 1996) from cattle and have sublethal effects on lymphocytes (Menge et al 2004;Ferens and Hovde 2007) and fibroblasts (Ferens and Hovde 2007). We recently demonstrated that EHEC O157:H7-secreted cytotoxins are toxic to epithelial cells from the jejunum and descending colon of cattle (Baines et al 2008). These results suggest that EHEC-secreted cytotoxins may have a role to play in EHEC O157:H7 colonization of intestinal tissues in cattle.…”

mentioning

confidence: 99%

“…Addition of the most active secreted cytotoxins from EHEC O157:H7 R318N to the IVOC adherence assays significantly increased the adherence of the most (R318N) and least (H4420N) virulent EHEC O157:H7 strain to intestinal tissues. The current study supports a role for EHEC O157:H7-secreted cytotoxins in enhancing EHEC O157:H7 colonization of intestinal tissues of cattle.Key words: Escherichia coli O157:H7, cattle, intestine, cytotoxins, colonization Baines, D., Masson, L. et McAllister, T. 2008. Les cytotoxines se´cre´te´es par Escherichia coli O157:H7 sont toxiques pour les ente´rocytes et favorisent la colonisation du je´junum et du coˆlon descendant des bovins par le microorganisme.…”

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Escherichia coli O157:H7-secreted cytotoxins are toxic to enterocytes and increase Escherichia coli O157:H7 colonization of jejunum and descending colon in cattle

Baines¹,

Masson²,

McAllister³

2008

Can. J. Anim. Sci.

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, T. 2008. Escherichia coli O157:H7-secreted cytotoxins are toxic to enterocytes and increase Escherichia coli O157:H7 colonization of jejunum and descending colon in cattle. Can. J. Anim. Sci. 88: 41Á50. Enterohemorrhagic Escherichia coli (EHEC) O157:H7-secreted cytotoxins are toxic to target cells and enhance colonization of intestinal tissues in disease-susceptible animals. It is unclear what role, if any, EHEC O157:H7-secreted cytotoxins play in the colonization of intestinal tissues of healthy reservoir animals. We previously reported that EHEC O157:H7 colonization sites were associated with focal hemorrhages in the jejunum and descending colon of persistent shedding cattle, suggesting a potential role for cytotoxins in EHEC O157:H7 colonization. We have used a traditional EHEC O157:H7 IVOC adherence assay and a novel lawn assay to examine the role of EHEC O157:H7-secreted cytotoxins in EHEC O157:H7 strain colonization of the jejunum and descending colon of non-persistent and persistent shedding cattle. Four EHEC O157:H7 strains that were previously reported to differentially colonize cattle produced cytotoxins that were differentially active against epithelial cells from the jejunum and descending colon. There was a relationship between EHEC O157:H7-secreted cytotoxin activity and strain adherence for epithelial cells from the jejunum and descending colon of cattle. There was also a greater susceptibility of epithelial cells from the jejunum and descending colon to EHEC O157:H7-secreted cytotoxins of persistent shedding cattle compared with non-persistent shedding cattle. Addition of the most active secreted cytotoxins from EHEC O157:H7 R318N to the IVOC adherence assays significantly increased the adherence of the most (R318N) and least (H4420N) virulent EHEC O157:H7 strain to intestinal tissues. The current study supports a role for EHEC O157:H7-secreted cytotoxins in enhancing EHEC O157:H7 colonization of intestinal tissues of cattle.Key words: Escherichia coli O157:H7, cattle, intestine, cytotoxins, colonization Baines, D., Masson, L. et McAllister, T. 2008. Les cytotoxines se´cre´te´es par Escherichia coli O157:H7 sont toxiques pour les ente´rocytes et favorisent la colonisation du je´junum et du coˆlon descendant des bovins par le microorganisme. Can. J. Anim. Sci. 88: 41Á50. Les cytotoxynes se´cre´te´es par la souche O157:H7 d'Escherichia coli ente´ro-he´morragique (EHEC) sont toxiques pour les cellules cibles et favorisent la colonisation des tissus intestinaux chez les animaux sensibles a`la maladie. Le roˆle e´ventuel des cytotoxynes de EHEC O157:H7 dans la colonisation des tissus intestinaux des porteurs sains n'est toutefois pas claire. Ante´rieurement, les auteurs avaient signale´que les sites colonise´s par EHEC O157:H7 e´taient associe´s a`des foyers d'he´morragie dans le je´junum et le coˆlon descendant des animaux excre´teurs, signe que les cytotoxynes libe´re´es par le microorganisme pourraient intervenir. Ils ont recouru a`une e´preuve classique d'adhe´rence IVOC pour EHEC O157:H7 ainsi ...

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Section: Ehec O157:h7 Secreted Cytotoxinsmentioning

confidence: 99%

Section: Ivoc Adherence Assaymentioning

confidence: 99%

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mentioning

confidence: 99%

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confidence: 99%

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Escherichia coli O157:H7-secreted cytotoxins are toxic to enterocytes and increase Escherichia coli O157:H7 colonization of jejunum and descending colon in cattle

Baines¹,

Masson²,

McAllister³

2008

Can. J. Anim. Sci.

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Escherichia coli O157:H7 Strain Origin, Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

Lowe

Baines

Selinger

et al. 2009

Appl Environ Microbiol

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Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine-and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 10 3 to 10 5 CFU and maximum colonization at 10 7 CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx 1 ) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx 2 ). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx 2 expression in modulating the amount of E. coli O157:H7 colonization of cattle.Enterohemorrhagic Escherichia coli O157:H7 is a bacterium that causes serious human disease outbreaks through the consumption of contaminated food or water (39). Mature cattle are considered the primary reservoir for E. coli O157:H7 and historically were reported to have no symptoms or pathologies (17,23,38); this was attributed both to a lack of receptors for a critical E. coli O157:H7 virulence factor, Shiga toxin 1 (Stx1 [29]), and to a differential expression of type III protein secretion system effector molecules such as EspA, EspD, and Iha (25,30) in cattle compared to humans. In 2008, it was established for the first time that E. coli O157:H7 causes mild to severe intestinal pathology in persistent shedding cattle (5,26) and that the secreted cytotoxins enhanced E. coli O157:H7 colonization of intestinal tissues of cattle (6). This suggested that cattle were susceptible to E. coli O157:H7 infection and that previously discounted virulence factors could influence the amount of colonization in cattle.Three distinct E. coli O157:H7 lineages have been identified based on the lineage specific polymorphism assay (LSPA-6) that suggests both the evolutionary history of the strain and their propensity to be present among animals, the environment, and clinical human isolates (21,22,24,33,40,42). Typically, two predominant lineages have been described, lineages I and II (22,40) and, more recently, intermediate lineages that have...

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Heterogeneity in enterohemorrhagicEscherichia coliO157:H7 fecal shedding in cattle is related toEscherichia coliO157:H7 colonization of the small and large intestine

2008

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In the last decade, Escherichia coli O157:H7 have emerged as important pathogens of the gastrointestinal tract of humans. Healthy cattle have been identified as the primary reservoir, however, the factors affecting heterogeneous E. coli O157:H7 fecal shedding are not fully understood. The aim of this study was to investigate the contribution of E. coli O157:H7 colonization of small and large intestinal sites to the heterogeneity of fecal shedding in cattle. There was a dose-dependant E. coli O157:H7 E318N colonization of duodenum, jejunum, ileum, cecum, ascending colon, spiral colon, descending colon, and the rectoanal junction in vitro with no difference in E. coli O157:H7 colonization of the rectoanal junction and other intestinal sites. There were 10-100 times greater E. coli O157:H7 colonization of intestinal sites from persistent shedding cattle compared with non persistent shedding cattle. Novel pathologies were associated with E. coli O157:H7 colonization sites in the small and large intestine. The first pathology, focal petechiae, was present throughout the intestinal tract of cattle that ceased shedding E. coli O157:H7 for 5-12 weeks or in the jejunum, ileum, cecum, and ascending colon of cattle shedding E. coli O157:H7 for 4-5 months. The second pathology, mucosal hemorrhages, was present in the same sites as the focal petechiae in cattle shedding for 5 months and these hemorrhages were in the final stages of repair. Several features of these hemorrhages support this conclusion including the brown appearance, low amount of classic E. coli O157:H7 induced A/E lesions, flattened epithelium, and blunted villi. Although mucosal hemorrhages were present in the jejunum, ileum, cecum, and ascending colon in cattle shedding for 4 months, many other pathologies were also present that were indicative of hemorrhagic enteritis as evidenced by the blood red appearance of hemorrhages, severe edema, and dark red erythema. Escherichia coli O157:H7 were associated with both pathologies suggesting it is the causative agent. The current study supports a relationship between the amount of E. coli O157:H7 colonization in intestinal sites and heterogeneous fecal shedding by cattle.

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A rapid, sensitive method for testing the activity of Escherichia coli 0157:H7 secreted cytotoxins against epithelial cells from the jejunum and descending colon of cattle

Cited by 6 publications

References 19 publications

Escherichia coli O157:H7-secreted cytotoxins are toxic to enterocytes and increase Escherichia coli O157:H7 colonization of jejunum and descending colon in cattle

Escherichia coli O157:H7-secreted cytotoxins are toxic to enterocytes and increase Escherichia coli O157:H7 colonization of jejunum and descending colon in cattle

Escherichia coli O157:H7 Strain Origin, Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

Heterogeneity in enterohemorrhagicEscherichia coliO157:H7 fecal shedding in cattle is related toEscherichia coliO157:H7 colonization of the small and large intestine

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