Development of a capture ELISA for the detection of antibodies to enteropathogenic Escherichia coli (EPEC) in rabbit flocks using intimin-specific monoclonal antibodies

Vandekerchove, Dominique; Kerr, P.; Callebaut, Alfons; Ball, H.J.; Stakenborg, Tim; Mariën, Jonas; Peeters, Jan

doi:10.1016/s0378-1135(02)00125-6

Cited by 5 publications

(3 citation statements)

References 18 publications

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“…Interest-ingly, animals aged Ͼ1 to 3 months showed the least frequent presence of both eae and stx; in contrast, those Ͼ3 to 6 months showed a generally higher prevalence. Intimin is known to be a strong immunogen (18), and it is classified into 10 types (21). Although several intimin type groups might generate immunological cross-reactions, E. coli strains possessing other intimin types can still infect animals.…”

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confidence: 99%

Prevalence and Characteristics of eae -Positive Escherichia coli from Healthy Cattle in Japan

Kobayashi

Miura²,

Hayashi

et al. 2003

Appl Environ Microbiol

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The prevalence of eae-positive Escherichia coli (eaeEC) in Japan was examined using rectal stool samples taken from 35 calves less than 1 month old, 107 calves more than 1 to 3 months old, 88 heifers more than 3 to 6 months old, 214 heifers over 6 months old, and cows from 95 farms. Screening with eae PCR revealed the prevalence to be, with increasing age, 31.4, 8.4, 26.1, and 14.5%, respectively. Of 51 selected eaeEC strains, more than 40% were serotyped as O26, O103, O111, O145, or O157, which are frequently detected as enterohemorrhagic E. coli types. Four strains were identified as recently reported intimin types , , and .Enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and attaching and effacing E. coli (AEEC) are food-borne pathogens that can cause diarrhea in humans (7,11,15). These pathogenic E. coli types often possess genes coding for Shiga toxins (stx genes) and for intimin (eae), an outer membrane protein. E. coli strains with stx genes are called Shiga toxin-producing E. coli (STEC). Cattle are considered to be the main reservoir of STEC strains, including EHEC strains (2, 3). STEC strains are classified into more than 200 O serotypes (4, 16); however, the majority of outbreaks and/or sporadic cases of hemorrhagic colitis and hemolyticuremic syndrome in humans have been caused by the members of only a few serogroups, such as O26, O111, and O157 (10,17,19). Since the strains of these limited O serogroups almost all possess eae (3, 9), this gene may be a more useful target than the stx genes for screening EHEC strains in cattle fecal samples.We used PCR to investigate the prevalence of eae-positive E. coli in cattle feces and genetically characterized the intimin types found, as well as various virulence genes seen in the isolated strains.E. coli O157:H7 strain ATCC 35150 (American Type Culture Collection, Manassas, Va.) was used as the positive control for stx 1 , stx 2 , eae, and intimin type ␥ (intimin ␥). The E. coli strains JS144, 166, VR299-2, and EPEC108, which were used as positive-control strains for intimins ␣, ␤, and ε and bundleforming pilus (bfp), respectively, were derived from the stock culture collection of the National Institute of Animal Health, Tsukuba, Japan. A total of 444 rectal stool grab samples were collected from healthy dairy cattle (35 calves under 1 month old, 107 calves more than 1 to 3 months old, 88 heifers more than 3 to 6 months old, 214 heifers more than 6 months old, and cows) on 95 farms located in the western and central parts of Japan between May and November 2001. All rectal stool samples were sampled by veterinarians at regional governmental animal hygiene centers. The samples were placed in cool boxes (4 to 8°C) and taken to the laboratory for immediate processing (usually within 24 h). Each sample of 1 g of rectal stool was enriched in 19 ml of Trypticase soy broth (Eiken, Tokyo, Japan) at 37°C for 18 h. Ten microliters of the Trypticase soy broth culture was inoculated onto MacConkey agar (MAC; Eiken). The MAC plates were incubated...

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Prevalence and Characteristics of eae -Positive Escherichia coli from Healthy Cattle in Japan

Kobayashi

Miura²,

Hayashi

et al. 2003

Appl Environ Microbiol

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“…Intimin has been successfully used in immunological-based assay development [23–26]. The variable 3’-region of Intimin has been used to confirm eae types and subtypes [3–5].…”

Section: Discussionmentioning

confidence: 99%

“…Using published methods [16, 23, 30, 33] with some modifications, we diluted original solid samples to 1:1 (v/v) mixtures, centrifuged them at 500 rpm to remove impurities, and then centrifuged supernatant at 16,000 rpm to create a bacterial pellet for selective enrichment. For liquids, filtered membranes were selectively enriched and optimized enrichment procedures increased the sensitivity of the DAS-ELISA.…”

Section: Discussionmentioning

confidence: 99%

Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1

Zhang

et al. 2016

PLoS ONE

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Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC’s intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples.

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Bacterial Diseases

DeLong

2012

The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents

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Development of a capture ELISA for the detection of antibodies to enteropathogenic Escherichia coli (EPEC) in rabbit flocks using intimin-specific monoclonal antibodies

Cited by 5 publications

References 18 publications

Prevalence and Characteristics of eae -Positive Escherichia coli from Healthy Cattle in Japan

Prevalence and Characteristics of eae -Positive Escherichia coli from Healthy Cattle in Japan

Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1

Bacterial Diseases

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