Pathogenic Shiga toxin-producing Escherichia coli in the intestine of calves

The purpose of this study was to compare the pathological effects of Shiga toxin-producing Escherichia coli (STEC) that vary in their association with bovine and human disease. Shiga toxin-producing E. coli of serotypes associated with both dysentery in calves and hemolytic uremic syndrome (HUS) in humans (O5:H-, O26:H11, O111:H-, O113:H21) were compared with O157:H7 STEC, which are associated with HUS in humans but not with disease in calves. The STEC were administered orally to 80 day-old chicks and into ligated loops in the ileum and colon of four 2- to 6-day-old calves. Examination of the ceca of the chickens 10 d postchallenge showed no adherence or tissue abnormality for any isolate. The calves were euthanized 8 to 10 h postinoculation, and sections of the intestinal loops were examined by light microscopy, transmission and scanning electron microscopy, and immunohistochemistry. All strains showed consistent focal adherence associated with mild lesions in the colon. Attaching and effacing lesions were observed with the eae-positive strains. Ileal lesions were similar to the colonic ones but were sometimes severe, with marked polymorphonuclear leukocyte proliferation in the lamina propria. It is concluded that chickens were unsuitable for studying interaction of STEC with the intestine and that there was no difference in the interaction of the ligated calf intestine with STEC of serotypes associated with disease in calves compared with O157:H7 STEC.     

Shiga toxin-producing Escherichia coli in the feces of Alberta feedlot cattle

Shiga toxin-producing Escherichia coli (STEC) are a public health concern. Bacterial culture techniques commonly used to detect E. coli O157:H7 will not detect other STEC serotypes. Feces from cattle and other animals are a source of O157:H7 and other pathogenic serotypes of STEC. The objective of this study was to estimate the pen-level prevalence of Shiga toxins and selected STEC serotypes in pre-slaughter feedlot cattle. Composite fecal samples were cultured and a polymerase chain reaction (PCR) was used to detect genes for Shiga toxins (stx1 and stx2) and genes for O157:H7, O111:H8, and O26:H11 serotypes. Evidence of Shiga toxins was found in 23 pens (92%), O157:H7 in 2 (8%), O111:H8 in 5 (20%), and O26:H11 in 20 (80%) of the 25 pens investigated. Although pen-level prevalence estimates for Shiga toxins and non-O157 serotypes seem high relative to O157:H7, further effort is required to determine the human health significance of non-O157 serotypes of STEC in feedlot cattle.     

产志贺毒素大肠杆菌检测技术研究进展

产志贺毒素大肠杆菌(Shiga toxin-producing Escherichiacoli. STEC)是一种重要的人畜共患病病原菌,可以引起人类水样腹泻、岀血性肠炎等轻度肠不适,严重时导致溶血性尿毒综合征(Hemolytic uremic syndrome, HUS)、终末期肾病(End stage renal disease, ESRD)甚至死亡。有研究显示,每年全球范围内估计感染致病型STEC造成超过2800000例人类急性疾病,死亡230例[1].鉴于STEC对全球公共安全的重要影响,对其高效检测和鉴定至关重要.     

Detection and characterization of Shiga toxin-producing Escherichia coli in feral pigeons

Escherichia coli strains producing a variant of Shiga toxin 2 (Stx2), designated Stx2f, have been recently described in the stools of feral pigeons. During 1997-1998, 649 pigeons were trapped and examined in three different squares of Rome. Stool samples were collected from each bird and enrichment cultures were examined for the presence of Stx by the vero cell assay. Stx-producing E. coli (STEC) were isolated from the positive cultures and characterized by serotyping and PCR analysis of stx and other virulence-related genes. Stx was detected in 10.8% of the stool enrichment cultures. The percentage of positive birds did not differ significantly for the three flocks considered and the season of sample collection. Conversely, STEC carriage was significantly more frequent in young than in adult birds (17.9 versus 8.2%). None of the birds examined showed signs of disease. STEC strains were isolated from 30 of 42 Stx-positive cultures examined. All the strains produced Stx2f, and most of them possessed genes encoding for intimin and the cytolethal distending toxin (CLDT). Six serogroups were identified, but most of the isolates belonged to O45, O18ab, and O75. Molecular typing indicated that most of the isolates within a flock were clonally-related. This work confirms that pigeons represent a natural reservoir of STEC strains characterized by the production of the toxin variant Stx2f, and by the frequent presence of eae and cldt genes. Further work is needed to clarify whether these STEC may represent a cause of avian disease or even a potential health hazard for humans.     

The occurrence of Shiga toxin-producing Escherichia coli in humans and animals

Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause haemorrhagic colitis and the diarrhoea-associated form of the haemolytic-uraemic syndrome in humans. The main cause of STEC infections in humans is the consumption of contaminated food. Both sporadic cases and outbreaks of STEC infections are mainly associated with the consumption of undercooked (minced) beef (mainly hamburgers) and unpasteurized milk. Therefore cattle are regarded as the primary reservoir of STEC. In this article the occurrence of STEC infections in humans and the occurrence of STEC in food and food-producing animals in the Netherlands are discussed, followed by a brief discussion of possible ways to prevent STEC infections in humans.     

Detection and characterization of Shiga toxin-producing Escherichia coli in captive non-domestic mammals

Shiga toxin producing-Escherichia coli (STEC) is an important emerging pathogen, and ruminants are recognized as their main natural reservoir. The aim of this work was to establish the frequency of STEC in non-domestic mammals of the Zoo and Botanical Garden of La Plata City, Argentina, and to pheno-genotypically characterize STEC isolates. By polymerase chain reaction (PCR), Shiga toxin (stx) gene sequences were detected in 50.8% of 65 fecal samples. Twenty-five STEC strains were isolated from 38.5% of the Zoo's animals. Ten species of order Cetartiodactyla and one species of order Rodentia were recognized as new STEC carriers. STEC strains belonged to 7 different serotypes including new serotypes O12:H25 and O13:H6. Serotype O146:H28, previously associated with human infections, represented 24% of STEC isolates. The most frequent Shiga toxin identified were type 1c and type 2c. Nineteen strains were positive for iha gene, 8 strains were positive for ehxA gene. Moreover, all strains were positive for lpfAO113 and negative for rfbO157, eae, saa, lpfAO157/OI-141, lpfAO157/OI-154, efa1, and toxB genes. Results obtained by XbaI-pulsed-field gel electrophoresis (XbaI-PFGE) confirmed the transmission of STEC strains among different animal species and suborders. In addition, we observed a potential association between STEC-harboring animal and factors such as belonging to order Cetartiodactyla, living in a pit, and belonging to a non-autochthonous species. This is the first work developed with zoological mammals and STEC in Argentina.     

Vascular ultrastructure and DNA fragmentation in swine infected with Shiga toxin-producing Escherichia coli

Shiga toxins (Stx) produced by Escherichia coli cause systemic vascular damage that manifests as edema disease in swine and hemolytic uremic syndrome in humans. In vitro, Stx inhibit protein synthesis and, depending on circumstances, induce necrosis, apoptosis, or both. The mechanism of in vivo Stx-mediated vascular damage is not known. The ability of Stx to cause apoptosis of vasculature in vivo was studied in pigs with edema disease that was produced by oral inoculation with Stx-producing E. coli. Arterioles of ileum and brain were evaluated by terminal dUTP nick-end labeling (TUNEL) assay for DNA fragmentation in myocytes (10 infected pigs, 5 control pigs) and by transmission electron microscopy for ultrastructural changes characteristic of apoptosis (17 infected pigs, 8 control pigs). In comparison with controls, increased numbers of TUNEL-positive arterioles were detected in 6/10 (60%) subclinically affected pigs 14-15 days after inoculation. Ultrastructurally, lesions in myocytes consisted of lysis (necrosis), with cytoplasmic debris and nuclear fragments contained between intact basement membranes. Endothelial cell changes ranged from acute swelling to necrosis and detachment from basement membrane. Subclinically affected pigs (n = 14) tended to have changes predominantly in myocytes, whereas pigs with clinical illness (n = 3) more commonly had changes in endothelial cells. The arteriolar lesions and clinical signs of edema disease are attributed to the effects of Stx on vasculature. Therefore, our findings suggest that the Stx-induced arteriolar lesions seen in this study were primarily necrotic, not apoptotic. We suspect that necrosis was the principal cause of the DNA fragmentation detected.     

Molecular characterisation of Shiga toxin-producing Escherichia coli O157 strains isolated in Poland

Ten Escherichia coli O157 strains isolated from cattle and children in Poland were investigated by the use of molecular biological methods. All strains possessed the intimin and enterohaemolysin genes and harboured the genetic determinants for Stx2 toxin (five isolates), Stx1 toxin (two strains) or both (three isolates). The genetic relatedness of the strains was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with Xbal and Notl. Nine closely related RFLP patterns were observed. Comparison of bovine and human E coli O157 isolates based on the analysis of Xbal and Notl digested profiles showed that all strains belonged to one genetic cluster. These results indicate that cattle must be considered as a possible source of human E coli O157 infection in Poland.     

Serotypes of verotoxin-producing (Shiga toxin-producing) Escherichia coli isolated from healthy sheep

Using PCR techniques Shiga toxin-producing strains of Escherichia coli were isolated from the faeces of 45 out of 101 healthy sheep. These strains were serotyped and found to include O5:H-, O91:H- and O163:H19, which had previously been reported as being associated with human disease including haemolytic uraemic syndrome.     

A national surveillance of Shiga toxin-producing Escherichia coli in food-producing animals in Japan

To assess the public health risk, the prevalence and anti-microbial resistance of Shiga toxin-producing Escherichia coli (STEC) among food-producing animals were studied throughout Japan. Faecal samples were collected from healthy animals of 272 cattle, 179 pigs, and 158 broilers on 596 farms in all 47 Japanese prefectures. STEC were isolated from 62 (23%) cattle and 32 (14%) pig samples but from no chicken samples. Of the bovine isolates, 19 belonged to serotypes frequently implicated in human disease (O157:H7/non-motile (NM)/H not typeable, O26:NM/H11/H21/H not typeable, O113:H21, and O145:NM). The eae genes were observed in 37% of bovine isolates; among them one O145:NM and all four O157 isolates possessed eae-gamma1, and one O145:NM, one O103:H11, and all five O26 isolates possessed eae-beta1 gene. Among the swine isolates, stx2e were dominant, and serotypes frequently implicated in human diseases or eae-positive isolates were not observed. Bovine isolates showed less anti-microbial resistance, but six isolates of 26:NM/H11 and O145:NM were multi-resistant and may need careful monitoring. Swine isolates showed various resistance patterns; chloramphenicol resistance patterns were more common than in bovine isolates. This first national study of STEC in the Japanese veterinary field should aid our understanding of Japan's STEC status.     

Association between animal age and the prevalence of Shiga toxin-producing Escherichia coli in a cohort of beef cattle

Even with advancements in pre- and post-harvest food safety, Shiga toxin-producing Escherichia coli (STEC) still present challenges to human health. Since cattle are the primary reservoir for STEC, lowering the prevalence of this pathogen in farm animals may reduce STEC outbreaks in humans. However, because many of the factors that modulate the colonization and persistence of STEC in cattle remain unknown, reducing STEC in this host is challenging. In this study, we evaluated a cohort of beef cattle one to eleven years of age to determine the effect of animal age on the prevalence of STEC. During the first year of sample collection, heifers had significantly lower STEC prevalence than cows (37.5% vs. 70%). In the second year of sample collection, STEC prevalence peaked in cows that were two years of age and tended to decrease as animals became older. In addition, by studying a subset of the animals in both years, we observed an increase in STEC prevalence from 40.6% to 53.1% in heifers, whereas cows had a net decrease in STEC prevalence from 71.4% to 61.9%. The results from this study indicate that animal age is a significant factor that influences the prevalence of STEC in cattle. These findings have implications for the development of on-farm mitigation strategies by targeting animals with the highest risk of shedding; it could be possible to reduce pathogen transmission among cattle and prevent zoonotic or foodborne transmission to humans.     

Detection and characterisation of Shiga toxin-producing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants

Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this work was to establish the prevalence of non-O157 STEC in free-ranging wild ruminants in the Extremadura region of Spain and to characterise them phenogenotypically. Faecal samples were collected from 243 wild ruminants, including Cervus elaphus, Capreolus capreolus, Dama dama and Ovis musimon and were examined for STEC using both phenotypic (Vero cells) and genotypic (PCR and PFGE) methods.Shiga toxin-producing Escherichia coli were isolated from 58 (23.9%) of the samples and a total of 65 isolates were characterised. A PCR method indicated that 11 (16.9%) strains carried the stx₁ gene, 44 (67.7%) carried the stx₂ gene and 10 (15.4%) carried both these genes. The ehxA gene was detected in 37 (57%) of the isolates but none contained either the eae or saa genes. The isolates were from a total of 12 ‘O’ serogroups, although 80% were restricted to the O2, O8, O128, O146, O166 and O174 serogroups. The most commonly isolated STEC bacteria, which were from the O146 serogroup, exhibited a high degree of polymorphism as indicated by PFGE. Shiga toxin-producing Escherichia coli isolates of serogroups O20, O25, O166, O171, O174 and O176 had not previously been found in wild ruminants. This is the first study to confirm that wild ruminants in Spain are a reservoir of STEC and are thus a potential source of human infection.     

Frequency of Shiga toxin-producing Escherichia coli in cattle at a breeding farm and at a slaughterhouse in Japan

To investigate the frequency of Shiga toxin-producing Escherichia coli (STEC) infected calves at a breeding farm and cattle at a slaughterhouse in Tohoku area of Japan, the polymerase chain reaction (PCR) was used for detection of genes for Shiga toxin(s). The fecal samples from a total of 204 calves and 306 cattle were examined. The prevalence rates in calves less than 2 months of age, cattle 2-8 months of age, and adults greater than 1 year of age were 39.4, 78.9, and 40.8%, respectively. Detection frequency of STEC in the fecal specimens from calves aged 0-8 months was not different among the breeds of cattle (Holstein: H, Japanese black cattle: B, and F1: HxB). On the other hand, for calves over 12 months of age, the frequency of STEC in Japanese black cattle and F1 were significantly higher than in Holstein cattle. Serogroups of STEC usually identified in human cases of food poisoning (O157, O26, and O111) were not frequently found in the feces of the cattle.     

Prevalence and characterization of Shiga toxin-producing Escherichia coli O157 and O26 in beef farms

Rectal content grab samples were collected from 2436 beef cattle reared on 406 beef farms in Japan between November 2007 and March 2008. STEC strains O157 and O26 were isolated from 110 (27.1%) and 7 (1.7%) farms, respectively. Farms that tested positive for STEC O157 were located in 35 out of all 47 Japanese prefectures. This indicates that STEC O157 strains are widespread on beef farms nationwide. Of the 2436 tested beef cattle, 218 (8.9%) and 10 (0.4%) had STEC strains O157 and O26 in the rectal content, respectively. The most common Shiga toxin genes detected in the isolated STEC O157 strains were: stx(2c) alone (32.1%), stx(2)/stx(2c) (27.2%), and stx(1)/stx(2) (21.8%). Almost all of the STEC O157 and STEC O26 strains expressed Shiga toxins (Stx). Most of the STEC O157 and STEC O26 strains possessed eaeA and EHEC-hlyA. These results strongly suggest that STEC strains O157 and O26 from beef cattle would be pathogenic to humans. Therefore, it is important to reduce STEC strains O157 and O26 in beef cattle in order to prevent foodborne disease caused by STEC. The presence of dogs and/or cats on a farm was significantly (P=0.02) associated with the prevalence of STEC O157. More research is needed to clarify the role of dogs and cats.     

Escherichia coli O157:H7 and other Shiga toxin-producing E. coli in white veal calves

The aims of the study were to determine the prevalence of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) and other Shiga toxin-producing E. coli (STEC) in feces of white veal calves in an operation in Ontario, to evaluate exposure of the calves to EHEC O157, and to investigate the milk replacer diet and antimicrobial resistance as factors that might influence the prevalence of EHEC O157. Feces from three cohorts of 20-21 calves were collected weekly for 20 weeks and processed for isolation of EHEC O157:H7 and detection of STEC by an ELISA. Exposure to EHEC O157 was also investigated by measuring IgG and IgM antibodies to the O157 lipopolysaccharide (O157 Ab) in sera by ELISA. The prevalences of EHEC O157 were 0.17% of 1151 fecal samples and 3.2% of 62 calves, and for STEC were 68% of 1005 fecal samples and 100% of 62 calves. Seroconversion to active IgG and IgM O157 Ab responses in some calves was not associated with isolation of EHEC O157. The milk replacer contained low levels of antibodies to EHEC antigens and without antimicrobial drugs, it did not inhibit the growth of EHEC O157 in vitro. Two E. coli O157:H7 that were isolated were totally drug sensitive whereas 60 commensal E. coli isolates that were examined were highly resistant. Antibodies in milk replacer that might be protective in vivo, and susceptibility to antimicrobial agents in the milk replacer may contribute to the low prevalence of EHEC O157 in white veal calves.     

Cryptosporidium and its potential as a food-borne pathogen

Cryptosporidium species are intestinal protozoan parasites and are excreted in animal feces as stable oocysts. Cryptosporidium has now been detected in the feces of a wide range of ruminant and non-ruminant farmed animals, wild animals, domestic pets and birds and the parasite appears to be well adapted to survive and persist in feces for extended periods, ranging from several weeks to many months. Because of this persistence, these materials are important as potential vehicles of transmission within herds, farms, the water chain, the fresh food chain, and the wider environment. Appropriate handling of animal waste is necessary to control spread of this pathogen and to limit the significant risks of human infection. While water is a well-recognized vector of Cryptosporidium, it has only recently emerged that food may play a more significant role than previously realized in the transmission of the Cryptosporidium to humans. In the last 3-5 years, research efforts have been directed both at the development of suitable methods for isolation and detection of the parasite in foods and at the application of these methods to assess the prevalence and persistence of the parasite in a range of foods. Additionally, molecular subtyping methods have been used to establish the transmission routes of the parasite. This paper summarizes the general biology of Cryptosporidium and overviews the current research on C. parvum in the food chain. The risks posed by certain foods, such as salad/vegetable crops and beef, are discussed and control measures which may be useful in the farm-to-fork chain for these products are described.     

Long-term survival of Shiga toxin-producing Escherichia coli in cattle effluents and environment: an updated review

Shiga toxin-producing Escherichia coli (STEC) are one of the most important emergent foodborne pathogens. STEC are common as colonizers in the intestine of healthy cattle and are spread into the environment by fecal shedding or following the surface application of farm effluent on soil. The bacteria can be transmitted to humans through food, such as inadequately cooked ground beef or unpasteurized milk. During the last decade, a wide variety of environmentally related exposures have emerged as new routes of transmission. Major outbreaks due to the consumption of raw fruits and vegetables or accidental ingestion of soil or water contaminated by STEC have been increasingly reported. STEC survival in cattle effluents, soil, plants and water is discussed in the light of new knowledge regarding both biotic and abiotic factors which may affect their survival or enhance their dissemination in the environment. The ability to persist in cattle production environments contributes to the contamination and recontamination of cattle, as well as for human infection. Consequently, effective control strategies must be considered on cattle farms, in order to limit entry of STEC cells into the environment.