Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR

Wang, Weilan; Zijlstra, R. T.; Gänzle, Michael G.

doi:10.1186/s12866-017-1023-5

Cited by 21 publications

(13 citation statements)

References 54 publications

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“…DNA concentration and quality were checked using NanoDrop ND-2000 spectrophotometer system (Fisher Scientific, USA). Quantification of E. coli and enterotoxins (STa, STb, LT) was conducted using real-time PCR, according to a method modified from Yang et al [34] and Wang et al [2]. Primers used for amplification of E. coli and enterotoxins in digesta samples were referenced from a previous study [34, 35] and listed in Table 3.…”

Section: Methodsmentioning

confidence: 99%

“…The pathogenesis of E.coli is mainly related to the adhesion of bacteria to the mucus of small intestine using surface proteins known as fimbriae [1]. In addition, ETEC strains can release cytotoxic enterotoxins, including heat-labile (LT) or two heat-stable entertoxins (STa, STb) into intestinal lumen, inducing diarrheal response [2]. It has been reported that K88+ ETEC are responsible for more than half of the piglet mortality each year worldwide [3, 4], leading to significant economic losses [5].…”

Section: Introductionmentioning

confidence: 99%

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Protective effect of chicken egg yolk immunoglobulins (IgY) against enterotoxigenic Escherichia coli K88 adhesion in weaned piglets

Wang

et al. 2019

BMC Vet Res

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Background Enterotoxigenic Escherichia coli K88 ( E. coli K88) are considered as a major cause of diarrhea and death in newly weaned piglets. Oral passive immunization with chicken egg yolk immunoglobulins (IgY) have attracted considerable attention for treatment of gastrointestinal infection due to its high specificity. In this study it was estimated the protective effect of anti-K88 fimbriae IgY against E. coli K88 adhesion to piglet intestinal mucus in vitro and to investigate the potential use of IgY for controlling E. coli -induced diarrhea in weaned piglets in vivo. Results E. coli K88 was incubated with IgY for 24 h, and the bacterial growth profiles showed that specific IgY with a concentration higher than 5 mg/mL was observed to significantly inhibit the growth of E. coli K88 compared to nonspecific yolk powder in a liquid medium. Moreover, pretreatment with 50 mg/mL of IgY was found to significantly decrease the adhesion ability of E. coli K88 to porcine jejunal and ileal mucus, further supported by the observations from our immunofluorescence microscopic analysis. In vivo, administration of IgY successfully protected piglets from diarrhea caused by E. coli K88 challenge. Additionally, IgY treatment efficiently alleviated E. coli -induced intestinal inflammation in piglets as the gene expression levels of inflammatory cytokines TNF-α, IL-22, IL-6 and IL-1β in IgY-treated piglets remained unchanged after E. coli K88 infection. Furthermore, IgY significantly prevented E. coli K88 adhering to the jejunal and ileal mucosa of piglets with E. coli infection and significantly decreased E. coli and enterotoxin expression in colonic contents. Conclusion Outcome of the study demonstrated that IgY against the fimbrial antigen K88 was able to significantly inhibit the growth of E. coli K88, block the binding of E. coli to small intestinal mucus, and protect piglets from E. coli -induced diarrhea. These results indicate that passive immunization with IgY may be useful to prevent bacterial colonization and to control enteric diseases due to E. coli infection. The study has great clinical implication to provide alternative therapy to antibiotics in E coli induced diarrhea. Electronic supplementary material The online version of this article (10.1186/s12917-019-1958-x) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Protective effect of chicken egg yolk immunoglobulins (IgY) against enterotoxigenic Escherichia coli K88 adhesion in weaned piglets

Wang

et al. 2019

BMC Vet Res

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“…E. coli can adhere to the intestinal epithelial cells and elaborate enterotoxins (LT, STa or STb). This induces diarrhoea and intestinal inflammation (Heo et al, ; Wang et al, ). In the experiments described here, dietary supplementation of antibiotics or E. coli K88‐specific IgY had no effect on ADG or ADFI compared to the control group.…”

Section: Discussionmentioning

confidence: 99%

“…DNA concentration and quality were checked using NanoDrop ND‐2000 spectrophotometer system (Fisher Scientific) prior to the samples being adjusted to a concentration of 10 ng/µl. Enterococcus , E. coli , Lactobacillus , Clostridium , Bifidobacterium and enterotoxins quantifications were conducted using real‐time PCR, according to the methods described in Wang, Zijlstra, and Ganzle (). Results were normalized total bacteria expression and relative fold changes calculated by the 2 −ΔΔCT method.…”

Section: Methodsmentioning

confidence: 99%

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Effect of chicken egg yolk immunoglobulins on serum biochemical profiles and intestinal bacterial populations in early‐weaned piglets

Tan

et al. 2019

Animal Physiology Nutrition

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This study was conducted to test the hypothesis that dietary supplementation with anti‐E. coli, chicken egg yolk immunoglobulins (IgY), may affect early weaned piglet (EWP) intestinal functions and enteric micro‐organisms. One hundred and forty‐eight ([Landrace × Yorkshire] × Duroc) piglets, weaned at age day 21, were randomly assigned to receive one of three diets for 14 days. Treatment group one (control group) was fed the base diet. Treatment group two (antibiotics group) was fed the base diet which was supplemented with 100 ppm colistin sulphate and 15 ppm enramycin; treatment group three (IgY group) was fed the base diet which was supplemented with 500 mg/kg anti‐E. coli IgY. The study evaluated the effects on EWPs of IgY on growth, serum biochemical, inflammatory profiles and also digestion content intestinal bacterial populations. Results showed no significant difference in diarrhoea rates between IgY‐fed EWPs and antibiotic‐treated EWPs. Serum biochemical analysis showed that EWPs fed an IgY‐containing diet had both lower (p < 0.05) cholesterol and low‐density lipoprotein compared to antibiotic‐treated EWPs. Escherichia coli populations measured in IgY‐fed EWP ileal contents, compared to the control group, were significantly reduced (p < 0.05). Enterococcus, Lactobacillus, Clostridium and Bifidobacterium populations were unaffected by the IgY treatment. Larger (p < 0.05) Enterococcus populations and lower (p < 0.05) expression levels of heat‐stable enterotoxin b (STb) were observed in IgY‐fed EWP caecal digesta compared to the control group. Enteric Lactobacillus significantly decreased (p < 0.05) in EWPs fed antibiotics while it was unaffected by IgY treatment. Dietary supplementation with anti‐E. coli IgY has the potential to suppress enteric E. coli growth, but not Lactobacillus, Clostridium and Bifidobacterium. This promotes and maintains a healthy EWP intestinal environment. These findings suggest that IgY may be used as an alternative to antibiotics in EWP diets.

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Effective Characterization of DNA Ligation Kinetics by High‐Resolution Melting Analysis

et al. 2019

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High‐resolution melting (HRM) analysis has been improved and applied for the first time to quantitative analysis of enzymatic reactions. By using the relative ratios of peak intensities of substrates and products, the quantitativity of conventional HRM analysis has been improved to allow detailed kinetic analysis. As an example, the ligation of sticky ends through the action of T4 DNA ligase has been kinetically analyzed, with comprehensive data on substrate specificity and other properties having been obtained. For the first time, the kinetic parameters (kobs and apparent Km) of sticky‐end ligation were obtained for both fully matched and mismatched sticky ends. The effect of ATP concentration on sticky‐end ligation was also investigated. The improved HRM method should also be applicable to versatile DNA‐transforming enzymes, because the only requirement is that the products have Tm values different enough from the substrates.

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Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR

Cited by 21 publications

References 54 publications

Protective effect of chicken egg yolk immunoglobulins (IgY) against enterotoxigenic Escherichia coli K88 adhesion in weaned piglets

Protective effect of chicken egg yolk immunoglobulins (IgY) against enterotoxigenic Escherichia coli K88 adhesion in weaned piglets

Effect of chicken egg yolk immunoglobulins on serum biochemical profiles and intestinal bacterial populations in early‐weaned piglets

Effective Characterization of DNA Ligation Kinetics by High‐Resolution Melting Analysis

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