Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins

Cattle are an important reservoir of Escherichia coli O157:H7 leading to contamination of food and water, and subsequent human disease. This pathogen colonizes its hosts by producing several proteins such as Tir and EspA that are secreted by a type III secretion system. These proteins play a role in colonization of the intestine, suggesting that they might be useful targets for the development of a vaccine to reduce levels of this organism in cattle. Vaccination of cattle with proteins secreted by E. coli O157:H7 significantly reduced the numbers of bacteria shed in feces, the numbers of animals that shed, and the duration of shedding in an experimental challenge model. Vaccination of cattle also significantly (P=0.04) reduced the prevalence of E. coli O157:H7 in a clinical trial conducted in a typical feedlot setting. This strategy suggests it is possible to vaccinate cattle to decrease the level of E. coli O157:H7 shedding for the purpose of reducing the risk of human disease.     

Fecal prevalence of Escherichia coli O157, Salmonella, Listeria, and Bacteriophage Infecting E. coli O157:H7 in feedlot cattle in the Southern Plains region of the United States

Escherichia coli O157:H7, Salmonella, and Listeria are foodborne pathogens of critical importance that often colonize cattle. E. coli O157:H7 can be specifically killed by lytic bacteriophage, and lytic bacteriophage treatment has been suggested as a pre-harvest intervention strategy to reduce foodborne pathogens in cattle. To date, no systematic approach to determine the incidence of E. coli O157:H7-infecting lytic bacteriophage has been published. Therefore, the current study was designed to determine (1) the incidence of E. coli O157, Salmonella spp., and Listeria and (2) the incidence of E. coli O157:H7-infecting bacteriophage in the feces of feedlot steers in commercial feedlots in the United States. Fecal samples (n=60) were collected from four feedlots in two Southern Great Plains states (total (n=240 fecal samples). Salmonella and E. coli O157:H7 were found in 3.8% and 11.7% of the fecal samples, respectively. Bacteriophage targeting E. coli O157:H7 were found in all four feedlots, in 15% of the individual fecal samples, and in 55% of the cattle pens. Our results indicate that such bacteriophage are widespread in feedlot cattle, suggesting that further research into the ecological role of bacteriophage in the gastrointestinal tract is needed.     

Escherichia coli O157:H7 diarrhoea associated with well water and infected cattle on an Ontario farm.

A 16-month old female child living on an Ontario dairy farm was taken to hospital suffering from bloody diarrhoea. Escherichia coli O157:H7 was isolated from her stool. Initial tests of well water samples were negative for E. coli by standard methods but culture of selected coliform colonies on sorbitol-MacConkey agar led to isolation of E. coli O157:H7. E. coli O157:H7 was also isolated from 63% of cattle on the farm. The E. coli O157:H7 isolates from the child, the water and the cattle were phage type 14, produced verotoxins 1 and 2, and were highly related on analysis by pulsed field gel electrophoresis. The child did not have known direct contact with the cattle and did not consume unpasteurized milk. Hydrogeological investigation revealed the design and location of the well would allow manure-contaminated surface water to flow into the well. This investigation demonstrates that cattle farm well water is a potential source of E. coli O157:H7 which may not be identified by standard screening for E. coli in water.     

Use of rope devices to describe and explain the feedlot ecology of Escherichia coli O157:H7 by time and place

Escherichia coli O157:H7 is an important pathogen of humans, and cattle populations serve as an important reservoir for human exposure. The organism is ubiquitous to feedlot cattle populations, although the nature of its occurrence is quite dynamic. Why E. coli O157:H7 varies by time and place in fed cattle is poorly understood. This study was designed to describe and explain the occurrence of E. coli O157:H7 by pen-level factors of time and place. From each pen, we cultured seven ropes placed within pens for cattle to rub and chew (ROPES), in order to classify the pens as high or low prevalence in longitudinal studies conducted during the summer and winter feeding periods of 2 full years. We observed differences in occurrence of ROPES-positive pens by season, weeks within season, and feedyard. ROPES-positive pens clustered temporally. Factors associated with ROPES-positive pen-weeks during both the summer and winter feeding periods were feedyard, prior 7-day mean air temperature, recovery of E. coli O157:H7 from the composite fecal sample, and recovery of E. coli O157:H7 from the water tank. Pens of summer-fed cattle were less likely to be ROPES-positive for E. coli O157:H7 if the ROPES were positive for Salmonella spp. The condition of the pen surface was associated with the likelihood for winter-fed pens of cattle to be ROPES-positive. We were able to monitor these pens of cattle using ROPES at minimal cost and without disturbing individual cattle. These observations improve our understanding of the ecology of E. coli O157:H7 in fed cattle, and also illustrate the importance of designing and analyzing observational studies and clinical trials to account for time- and place-dependent variables that affect the probability of detecting E. coli O157:H7.     

Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts

Escherichia coli O157:H7 can live undetected in the gut of food animals and be spread to humans directly and indirectly. Bacteriophages are viruses that prey on bacteria, offering a natural, nonantibiotic method to reduce pathogens from the food supply. Here we show that a cocktail of phages isolated from commercial cattle feces reduced E. coli O157:H7 populations in the gut of experimentally inoculated sheep. A cocktail of phages was used in order to prevent the development of resistance to the phages. In our first in vivo study we found that our cocktail of phages reduced E. coli O157:H7 populations in the feces of sheep (p < 0.05) by 24 hours after phage treatment. Upon necropsy, populations of inoculated E. coli O157:H7 were reduced by phage treatment in both the cecum (p < 0.05) and rectum (p < 0.1). In our second in vivo study, several ratios of phage plaque-forming units (PFU) to E. coli O157:H7 colony-forming units (CFU) were used (0:1, 1:1, 10:1, and 100:1 PFU/CFU) to determine the most efficacious phage dose. A 1:1 ratio of phage to bacteria was found to be more effective (p < 0.05) than either of the higher ratios used (10:1 or 100:1). Ruminal levels of E. coli O157:H7 were not significantly reduced (p > 0.10) in any of the studies due to relatively low inoculated E. coli O157:H7 ruminal populations. Our results demonstrate that phage can be used as a preharvest intervention as part of an integrated pathogen reduction scheme.     

Escherichia coli O157:H7 and Salmonella in water and soil from tall fescue paddocks

Six 1-ha paddocks of tall fescue (Festuca arundinacea) grazed by Gelbvieh x Angus heifers from 1 March to 21 June 2005 were used to determine the prevalence of Escherichia coli O157:H7 and Salmonella in water tanks, and standing water and surface soil surrounding water tanks in tall fescue paddocks grazed by cattle. Paddocks included two each of Kentucky-31 endophyte-infected tall fescue (E+), Jesup tall fescue with the AR542 endophyte strain (MaxQ), and HiMag tall fescue with strain 4 endophyte (HiMag4). Samples were collected weekly (6 June to 5 July) from water tanks (n = 30), and standing water (n = 18) and surface soil (n = 30) surrounding the water tanks in each paddock commencing 3 weeks prior to termination of grazing until 2 weeks after cattle removal. Prevalence of E. coli O157:H7 tended (p = 0.07) to be increased in standing water and surface soil surrounding water tanks when cattle were present. Presence of cattle resulted in muddy conditions surrounding water tanks. Consumption of E+ tall fescue did not influence (p > 0.10) the prevalence of E. coli O157:H7 or Salmonella in or around water tanks. Neither E. coli O157:H7 or Salmonella were detected in water tanks. Total percentage of standing water samples positive for E. coli O157:H7 was 27.8% and 5.6% for Salmonella. Escherichia coli O157:H7 (6.7%) and Salmonella (10%) also were detected in the surface soil surrounding the water tanks. We conclude that areas surrounding water tanks in tall fescue paddocks can be reservoirs of pathogenic bacteria.     

Escherichia coli: on-farm contamination of animals

Escherichia coli is one of the main inhabitants of the intestinal tract of most mammalian species, including humans, and birds. Shiga toxin-producing E. coli (STEC), also called verotoxinogenic E. coli, usually do not cause disease in animals but may cause watery diarrhoea, haemorrhagic colitis, and/or haemolytic uraemic syndrome in humans. Zoonotic STEC include the O157:H7 strains and, with increasing frequency, certain non-O157 strains. The importance of non-O157 zoonotic strains is probably underestimated as they have been less well characterised and are more difficult to detect in samples than O157:H7. Another large subset of STEC strains has been isolated from animals but has not, at the present time, been associated with disease in animals or humans. Cattle and other ruminants are the most important reservoir of zoonotic STEC, which are transmitted to humans through the ingestion of foods or water contaminated with animal faeces, or through direct contact with the infected animals or their environment. The main sources of STEC infection of cattle on-farm are the drinking water, the feed, and the immediate environment of the animal. Risk factors that have been identified for infection of animals with O157 STEC include age, weaning, movement of the animals, season, feed composition, and the ability of the bacteria to persist in the environment. On-farm control of the zoonotic risk of human infection with STEC should primarily target the main source of contamination: the animal reservoir. Various strategies to reduce intestinal colonisation of cattle by zoonotic STEC have been tried with varying results, including vaccination, treatment with probiotics, such as direct-fed microbials or competitive exclusion, administration of bacteriophages, and modification of the diet.     

Growth and survival of Escherichia coli O157:H7 during manufacture and storage of Indian cheese (paneer)

The growth and survival of Escherichia coli O157:H7 during manufacture and storage of Indian cheese or paneer was investigated. Pasteurized and raw milk that had been inoculated with 10(4) CFU/mL of E. coli O157:H7 cultures were used for manufacturing paneer samples which were vacuum-packaged and stored at 4 degrees C, 8 degrees C, and 28 degrees C. Survival and growth of E. coli O157:H7 in paneer samples was determined after every 4 h for up to 48 h. Escherichia coli O157:H7 could survive the manufacturing process of paneer and were present at the end of the storage period (at 28 degrees C) in significantly (p < 0.05) greater numbers than the initial inoculum. No significant difference in survival and growth were noticeable in paneer samples manufactured from raw milk. Though refrigeration (4 degrees C or 8 degrees C) effectively inhibited the growth of E. coli O157:H7, this pathogen could survive over a period of 48 h. Our observations suggest that unpasteurized or improperly pasteurized milk could be an important source for transmission of E. coli O157:H7 through paneer, and appropriate steps must be taken by government agencies to ensure consumer safety for this dairy product.     

应用免疫磁珠法首次在宝山区检出大肠杆菌O_(157):H_7

[目的 ]　了解宝山区是否存在大肠杆菌O157∶H7以及动物带菌和食品、水源的污染情况。　 [方法 ]　对采集的肉、乳制品、家禽、家畜粪便 ,快餐原料、各类水源等标本应用免疫磁珠法集菌进行E .coliO157∶H7分离和鉴定。　 [结果 ]　共采集家禽、家畜、肉乳制品、快餐原料、各类水源等标本 610份 ,检出E .coliO157∶H73株 ,总检出率为 0 .49%。其中以肉类制品检出率最高 ,为 1.3 7% ;其次为其它标本 ,检出率为 1.0 2 % ,家禽、家畜粪便 ,检出率为 0 .3 8%。　 [结论 ]　宝山区存在O157∶H7大肠杆菌感染爆发或流行的隐患     

The prevalence of Escherichia coli O157.H7 in dairy and beef cattle in Washington State.

Escherichia coli O157.H7 was found in 10 of 3570 (0.28%) faecal samples from dairy cattle in 5 of 60 herds (8.3%). Several tentative associations with manure handling and feeding management practices on dairy farms were identified. Faecal/urine slurry samples, bulk milk samples, and milk filters from dairy herds were negative for E. coli O157.H7. E. coli O157.H7 was also isolated from 10 of 1412 (0.71%) faecal samples from pastured beef cattle in 4 of 25 (16%) herds. The prevalence of E. coli O157.H7 excretion in feedlot beef cattle was 2 of 600 (0.33%). The identification of cattle management practices associated with colonization of cattle by E. coli O157.H7 suggests the possibility that human E. coli O157.H7 exposure may be reduced by cattle management procedures.     

上海市闵行区家禽畜大肠杆菌O157∶H7带菌状况监测

[目的 ]　了解闵行区家禽畜中大肠杆菌O15 7∶H7的带菌情况。　 [方法 ]　对采集的禽畜粪便应用免疫磁珠富集分离法进行大肠杆菌O15 7∶H7分离和鉴定。　 [结果 ]　 3 5 4份样品中检出 2 2份大肠杆菌O15 7∶H7,检出率为6.2 1% ,经鉴定均非产毒株。　 [结论 ]　闵行区家禽畜粪便中大肠杆菌O15 7∶H 7检出率较高 ,应引起重视     

Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.     

Evaluation of a multiplex real-time polymerase chain reaction for the quantification of Escherichia coli O157 in cattle feces

Cattle are asymptomatic reservoirs for Escherichia coli O157, a major foodborne pathogen. The organism generally colonizes the hindgut of cattle and is shed in the feces at low concentrations. The objective of this research was to evaluate a multiplex, real-time polymerase chain reaction (mqPCR) assay for quantification of E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets. Primer efficiency and analytical sensitivity of the assay were evaluated with a single or pooled (five strain) culture of E. coli O157. In pure culture, the minimum detection limit of the assay was 1.4×10(3) CFU/mL and 3.6×10(3) CFU/mL for the single and five-strain mixture of E. coli O157, respectively. Diagnostic sensitivity was analyzed using DNA extracted from cattle feces spiked with E. coli O157. In feces spiked with the pooled mixture of five E. coli O157 strains, the minimum detection limit was 3.6×10(4) CFU/g. We also evaluated the assay with feces from cattle experimentally inoculated with E. coli O157 by comparing the results to a culture-based method. For the majority of samples tested, the concentration of E. coli O157 detected by the real-time and culture methods was within one log difference. However, the assay could only be evaluated for cattle shedding high concentrations of E. coli O157. In conclusion, the mqPCR quantifying E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets may have use in detecting and quantifying super shedders, but is not applicable for quantification in animals shedding low concentrations (10(2) to 10(3) CFU/g feces).     

Thermal tolerance of acid-adapted and non-adapted Escherichia coli O157:H7 and Salmonella in ground beef during storage

Thermal tolerance of acid-adapted Escherichia coli O157:H7 or Salmonella in ground beef was evaluated during storage at 4 degrees C or -20 degrees C. Both pathogens were adapted to acidic conditions (pH approximately 4.6) by growing in tryptic soy broth supplemented with 1% glucose. A five-strain cocktail of E. coli O157:H7 or Salmonella was grown separately in TSB (pH approximately 6.6) and TSB + 1% glucose for 24 h at 37 degrees C to provide cells with or without acid adaptation. Irradiated ground beef was inoculated with either acid-adapted or non-adapted E. coli O157:H7 or Salmonella; the samples stored at 4 degrees C were subjected to heat treatment at 62 degrees C or 65 degrees C on days 1, 7, 14, 21, and 28, and the samples stored at -20 degrees C were subjected to heat treatment at 62 degrees C or 65 degrees C on days 1, 30, 60, 90, and 120. Decimal reduction time (D values) of the pathogens was determined as an indicator of thermal tolerance. Significantly higher D(62) values were observed on days 21 and 28 for non-adapted E. coli O157:H7 stored at 4 degrees C and on days 90 and 120 for non-adapted E. coli O157:H7 stored at -20 degrees C (P < 0.05). Higher D(62) values were observed on days 21 and 28 among non-adapted Salmonella strains stored at 4 degrees C and on day 28 for acid-adapted strains of Salmonella stored at 4 degrees C (P < 0.05). Higher D(62) values for acid-adapted strains of Salmonella stored at -20 degrees C were observed on days 30, 60, and 90 (P < 0.05), when while no differences were observed in the D(65) values of acid-adapted and non-adapted strains of E. coli O157:H7 and Salmonella throughout storage at both temperatures (P > 0.05). This suggests that acid adaptation of foodborne pathogens provides a certain level of protection against heat treatment at lower cooking temperatures, while at higher temperatures there were no observed differences between the sensitivity of acid-adapted and non-adapted strains in an actual food system over an extended period of refrigerated and frozen storage.     

Escherichia coli O157 in the rectoanal mucosal region of cattle

The rectoanal junction mucosal region is the site of colonization of Escherichia coli O157 in cattle. Our objective was to determine the genetic relatedness of E. coli O157 in the mucosa of the rectoanal junction to isolates from colon contents and feces. Colon contents and rectums were collected from cattle at harvest. Rectums were opened and feces were sampled with a cotton swab. The mucosa of the rectum was cleansed free of visible feces with water and saline. The region, 2 to 5 cm proximal to the rectoanal junction, was swabbed with a foam-tipped applicator and then incisions were made in this region and the submucosa was swabbed with an applicator. Isolation and identification of E. coli O157 was performed in accordance with well-documented methods. Prevalence of E. coli O157 in the colon contents, feces, rectal mucosa, and rectal submucosa was 21%, 29%, 54%, and 34%, respectively. Pulsed-field gel electrophoresis was used to compare clonal similarity among isolates from different sampling regions. Sixty-seven cattle had E. coli O157 isolated from the rectal mucosa swab and feces of which 82% were clonally similar (dice similarity >95%) within animal. Escherichia coli O157 isolates from feces and colon contents were similar in 76% of cattle, but E. coli O157 isolates from the rectoanal mucosal swab and colon contents were only similar in 61.4% of cattle. Our results suggest that E. coli O157 in the feces may be from two sources, colonized in the rectoanal mucosa or transient in the gastrointestinal tract.     

Citrus products decrease growth of E. coli O157:H7 and Salmonella typhimurium in pure culture and in fermentation with mixed ruminal microorganisms in vitro

Orange peel and orange pulp are by-products that are included in feedlot and dairy cattle diets because of their low cost and high nutritional quality. The antimicrobial activity of citrus oils has been reported previously. The present study was carried out to determine whether these citrus by-products exert antimicrobial effects on Escherichia coli O157:H7 and Salmonella Typhimurium populations that are found in cattle gastrointestinal tracts. The growth of pure cultures (n = 3) of E. coli O157:H7 and Salmonella Typhimurium were reduced (p < 0.05) by addition of 2% (w/v) orange pulp and orange peel. Ruminal fluid was collected from cattle (n = 2) and E. coli O157:H7 or Salmonella Typhimurium were added. The addition of orange pulp and peel to in vitro mixed ruminal microorganism fermentations (n = 3) demonstrated that both orange pulp and peel reduced E. coli O157:H7 and Salmonella Typhimurium populations at least 2 log(10) in mixed ruminal fluid fermentations. Addition of orange pulp reduced (p < 0.05) E. coli O157:H7 populations from 10(5) to 10(2) colony-forming units (CFU)/mL and Salmonella Typhimurium populations (p < 0.05) from 10(4) to 10(2) CFU/mL. These results indicate that orange pulp and/or peel included in ruminant diets could decrease ruminal populations of foodborne pathogenic bacteria. Further research is needed to determine whether the antimicrobial activity of orange products against E. coli O157:H7 or Salmonella Typhimurium is expressed in the lower gastrointestinal tract.     

Laboratory investigation of an E. coli O157:H7 outbreak associated with swimming in Battle Ground Lake,Vancouver, Washington

Escherichia coli O157:H7 has been associated with a number of waterborne outbreaks, but it has never been recovered from an implicated environment. This paper reports on an August 1999 outbreak of E. coli O157:H7 associated with swimming in Battle Ground Lake in Clark Country, Washington. E. coli O157:H7 was isolated from duck feces, as well as from two water samples. The authors used pulsed-field gel electrophoresis to compare these isolates with patient isolates for genetic homology. All the isolates yielded the same restriction fragment patterns. In addition, using polymerase chain reaction, the authors found patient isolates and environmental isolates to have the same virulence factors (Stx, eaeA, and hly).     

绿色荧光蛋白在大肠杆菌O157∶H7检测中的应用

目的　将绿色荧光特征引入大肠杆菌E coliO15 7∶H7,用于传统检测方法的改进及目的菌的应用研究。方法　将pGFP质粒转化E coliO15 7∶H7,构建一种工程菌 (E coliO15 7∶H7 pGFP) ,并进行了选择性培养基的筛选。以E coliO15 7∶H7 pGFP对鸡肉与牛奶等食品样品进行接种与回收试验 ,同时设定不同温度模拟食品储存的不同情况。结果　pGFP质粒可以在E coliO15 7∶H7菌株中稳定存在。将E coliO15 7∶H7 pGFP接种食品样品 ,以LBan培养基平板回收发现 :较高温度存放肉类与牛奶 ,其污染的E coliO15 7∶H7菌量可在 12h增加 35 0 0 0～ 2 0 0 0 0 0倍 ,而 4℃存放可以使污染菌量缓慢下降。结论　构建了一种具有紫外灯下发出绿色荧光与氨苄青霉素抗性等特性的重组菌株———O15 7∶H7 pGFP ,并设计了适合E coliO15 7∶H7 pGFP生长的选择性培养基———LBan。该重组菌株可应用于检测方法的改进及该菌特性的研究 ,是一种非常有用的工程菌     

A cluster of Escherichia coli O157: nonmotile infections associated with recreational exposure to lake water

OBJECTIVES: To identify cases and determine risk factors for an outbreak of Escherichia coli (E. coli) O157: nonmotile (NM) infections in children attending a summer day care program in California. METHODS: The authors conducted a retrospective cohort study; the cohort comprised first and second graders who attended the day care program during the last week in August 1999. Shiga toxin testing and molecular subtyping using pulsed-field gel electrophoresis were performed on isolates. Lake water, lake bottom sediment samples, and waterfowl feces from the lake environs were cultured for E. coli O157. RESULTS: Three cases of Shiga toxin-producing E. coli O157: NM infections with matching pulsed-field gel electrophoresis patterns and four probable cases were found. Children who swallowed more than a mouthful of water had a higher attack rate than those who swallowed less than a mouthful or none at all (43% vs. 10%, relative risk = 4.43, 95% confidence interval 1.12, 17.50). CONCLUSIONS: E. coli O157: NM infections were associated with swallowing water from a freshwater lake. Potential sources of contamination include feces from humans, cattle, or deer. This outbreak illustrates the value in screening patients with diarrhea for E. coli O157, submitting isolates to public health laboratories, and using molecular techniques to identify related cases. Outbreaks associated with contaminated freshwater could be averted by prevention and early detection of contamination.     

不同来源产志贺毒素大肠埃希菌分布特征

目的探讨不同标本中产志贺样毒素大肠埃希菌（STEC）的分布特征。方法采集动物粪便、肉类食品和排污口污泥样品，常规分离大肠埃希菌，血清学分型，PCR鉴定产志贺样毒素（stx1，stx2）菌株。结果293份标本中鉴定出8株STEC，1株为产志贺毒素O157：H7型，2株为不产志贺毒素O157：H7型，5株为产志贺毒素非O157：H7型。结论STEC存在于不同来源的标本中，菌株表型与毒力因子存在一定差异。