Shiga toxin-producing Escherichia coli and their bacteriophages as a model for the analysis of virulence and stress response of a food-borne pathogen

Enterohemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing Escherichia coli (STEC) that are able to cause serious food-borne intestinal diseases which can be followed in 5 to 15% by extraintestinal sequelae such as the hemolytic-uremic syndrome (HUS). One of the major pathogenicity factors of EHEC is the production of one or more Shiga toxins (Stx), which act as inhibitors of protein biosynthesis and have profound effects on the signal transduction and immunological response in eukaryotic cells. The stx genes are located in the genome of heterogeneous, lambdoid, functional or cryptic bacteriophages and are expressed during the phage life cycle. Due to the linkage between the phage life cycle and stx expression, STEC and their bacteriophages are useful as a model for the analysis of stress response and virulence of this food-borne pathogen.     

Emerging enterohaemorrhagic Escherichia coli, causes and effects of the rise of a human pathogen

Shiga toxin (Stx) [Verotoxin (VT)]-producing Escherichia coli (STEC), also called enterohaemorrhagic E. coli or VTEC are emerging zoonotic agents and became most important as human pathogens, particularly in the industrialized countries. Production of cytotoxins, also called Stx or VT, is the major pathogenicity determinant of STEC, which can cause life-threatening haemorrhagic diseases in humans. The spectrum of STEC phenotypes is diverse and domestic and wildlife animals constitute important reservoirs for these bacteria. STEC are spread from animal faeces to the environment, water and food. Ingestion of contaminated foodstuff and water, as well as contact with the environment, STEC-excreting animals or humans are the major sources of human infection. Economical changes in animal and food production, alteration of consumer habits and lack of specific immune response, particularly in urbanized populations, have contributed to the recent spread of STEC as a zoonotic agent. Supranational surveillance networks as well as national reference laboratories as sentinels play an important role in the prevention and control of STEC infections in humans. Development of new vaccines and probiotics may serve as future tools to control the spread of STEC in animals and humans.     

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.     

Enterohaemorrhagic Escherichia coli O26:H11/H-: a human pathogen in emergence

Enterohaemorrhagic Escherichia coli (EHEC) O26:H11 have emerged as the most important non-O157:H7 EHEC, with respect to their ability to cause diarrhoea and the haemolytic uraemic syndrome (HUS). HUS is a leading cause of acute renal failure in children, and is mainly caused by EHEC expressing Shiga toxins (Stx) 1 and/or 2. Since 1996, EHEC O26, which produce Stx2 only and appear to have enhanced virulence, have been increasingly isolated from HUS patients in Germany. In contrast, EHEC O26 found in cattle predominantly produce Stx1 as the sole Stx. Additional potential virulence factors of EHEC O26 include cytolysins (EHEC hemolysin), serine proteases (EspP), lymphotoxins (Efal) and adhesins (intimin). The genes encoding the virulence factors are located within pathogenicity islands (eae, efa1), bacteriophages (stx) or plasmids (EHEC-hlyA, espP). In addition, EHEC O26 possess, in contrast to other EHEC, the "high pathogenicity island" (HPI), which is also present in pathogenic Yersiniae.This island contains genes involved in the biosynthesis, regulation and transport of the siderophore yersiniabactin. Comparative genomic analyses between EHEC O26 and non-pathogenic E. coli, as well as investigations of mechanisms involved in the transfer of virulence genes, provide a deeper insight into the evolution of EHEC O26.These studies demonstrate how horizontal transfer of virulence genes, even from distantly related organisms, can lead in brief intervals to the rise of a highly virulent clone within a particular E. coli serotype.The classical bacteriological methods are no longer sufficient to determine the risk posed by EHEC O26. However, knowledge of the complete virulence profiles of these pathogens and understanding their stepwise evolution form a foundation for developing new strategies to prevent human infections and new methods for their laboratory diagnosis.     

Comparison of binding and effects of Escherichia coli Shiga toxin 1 on bovine and ovine granulocytes

Granulocytes play a pivotal role in the pathogenesis of Shiga toxin (Stx)-producing Escherichia coli (STEC) related diseases in humans. Granulocytes are attracted and activated by Stxs in the enteric mucosa and are believed to thereby contribute to the intestinal inflammation. Mature ruminants, the main reservoir hosts of STEC, do not develop pathological changes that can be attributed to the Stxs. To prove whether the latter phenomenon correlates with the inability of the Stxs to affect granulocytes of ruminants, we investigated the ability of Stx1 to bind to granulocytes of cattle and sheep and analysed the effects of Stx1 on viability, phagocytosis, and oxidative burst activity. Bovine granulocytes from blood and milk did not express Stx1-binding sites even after activation of the cells and also were resistant to Stx1. In contrast to bovine granulocytes, granulocytes of sheep constitutively expressed Stx1-receptors of the Gb(3)/CD77 type ex vivo and bound the recombinant B-subunit of Stx1 (rStxB1). Stx1 holotoxin induced apoptosis in ovine granulocytes after prolonged incubation (18h) but Stx1 only slightly altered the phagocytosis and oxidative burst activities. The rStxB1 had no effect on granulocytes of either species. While arguing in favour of our initial hypothesis, that granulocytes of both, cattle and sheep are not activated by Stxs, the results of our study are the first evidences for differences in the cellular distribution of Stx-receptors in species equally regarded as STEC carriers.     

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.     

Identification of virulence factors by multiplex PCR in Escherichia coli isolated from calves in Minas Gerais, Brazil

In this study, multiplex PCR was employed to investigate the virulence factors of Escherichia coli strains isolated from 60-day-old calves. Faecal samples were collected from 54 calves at 12 dairy farms in the state of Minas Gerais, Brazil. A total of 156 isolates were obtained after culture and microbiological isolation and were tested by multiplex PCR for the presence of genes encoding toxins (Stx1, Stx2 and STa) and adherence factors (intimin, F41 and F5). Seventy of 156 isolates were positive for at least one virulence factor: ten (14.3?%) from diarrhoeic animals and 60 (85.7?%) from healthy calves. The virulence markers identified were: Stx1 (82.8?%), eae (24.3?%), F41 (11.4?%), F5 (10?%), STa (4.28?%) and Stx2 (4?%). In diarrhoeic animals, Stx1 (70?%) and F41 (30?%) were identified, while Stx1 (83.3?%), eae (28.3?%), F41 (8.3?%), F5 (11.6?%), STa (5?%) and Stx2 (1.6?%) were detected in isolates from healthy calves. Mixed infections with pathotypes Shiga toxin-producing E. coli (STEC)/enteropathogenic E. coli, STEC/enterohaemorrhagic E. coli and STEC/other (eae/F5, Stx1/STa) were detected in five healthy calves. Pathogenic E. coli were identified in 59.26?% of all calves and on 75?% of the dairy farms studied, not only in diarrhoeic (five of six) but also in healthy calves (27 of 48), which demonstrates the importance of this agent in the aetiology of diarrhoea in calves in the state of Minas Gerais.     

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.     

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.     

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.     

Animal health and foodborne pathogens: enterohaemorrhagic O157:H7 strains and other pathogenic Escherichia coli virotypes (EPEC,ETEC, EIEC,EHEC)

The majority of interactions between microorganisms and animals are based on convenient relations for both of them. Symbiotic microorganisms, like intestinal microbiota, produce important vitamins for animals and protects them from putative pathogens. In general, for monogastric animals, the main contribution of intestinal microorganisms is to supply with growth factors the animal diet, and in some cases they are responsible for providing essential vitamins (e.g. vitamin K). Some particular and relatively few microbes like viruses, bacteria, fungi, protozoa and algae are responsible for animal illness. Because microorganisms are easily dispersed, display physiological diversity, and tolerate extreme conditions, they are ubiquitous and may contaminate and grow in many products, including food and raw materials. Foodborne diseases are caused by consumption of contaminated food or beverages. Many different disease-causing pathogens can contaminate food, so there are many different foodborne infections. In addition, poisonous chemicals and biological toxins can cause disease if they are present in food. To know how a particular disease is spreading is an important matter to take appropriate steps to stop it. For example Escherichia coli O157:H7 infections can spread through contaminated food (meat, vegetables, cheese, etc.), contaminated drinking water or juices, contaminated swimming water and from person to person. Among foodborne pathogens, the most frequently detected are bacteria, but also parasitic protozoa and worms, viruses, natural toxins and other pathogenic agents like prions are important agents for foodborne diseases. Particular pathogenic types of E. coli, classified by their specific pathogenic mechanisms (toxins, adhesins, invasiveness, etc.) are actually known as E. coli virotypes. Enterohaemorrhagic E. coli (EHEC), which constitute the main part of this review, were also named verotoxigenic E. coli (VTEC) or Shiga toxigenic E. coli (STEC). EHEC strains cause haemorrhagic colitis (HC), haemolytic uremic syndrome (HUS) and thrombotic thrombocytopaenic purpura (TP) in humans. They synthetize shigatoxins (verotoxins) which are potent cytotoxic substances, adherence factors and enterohaemolysin. EHEC are responsible for many outbreaks of bloody diarrhoea caused by contaminated foods: beef, milk, fruits, juice, water, etc. The most important serogroups among EHEC are O26, O111 and O157, being O157:H7 the most relevant serotype in foodborne outbreaks. The normal intestinal microflora of cattle was found to be the most relevant reservoir of EHEC strains.     

Activatable Shiga toxin 2d (Stx2d) in STEC strains isolated from cattle and sheep at slaughter

Shiga toxin producing Escherichia coli (STEC) harbouring the stx(2d-activatable) gene and expressing the mucus- and elastase-activatable phenotype have been associated with severe outcomes of human disease. However, there is limited data available on the occurrence of such strains in livestock reservoirs. In this study, we analyzed 11 STEC strains isolated from healthy cattle and sheep at slaughter that were originally detected to contain the stx(2c) allele, for the presence of the stx(2d-activatable) genotype. Ten of the eleven strains displayed the stx(2d-activatable) genotype as determine by PstI restriction fragment length polymorphism (RFLP) of 890-bp fragments of their stx genes. However, only in 6 of the 10 strains whose stx genes were sequenced, the presence of stx(2d-activatable) could be confirmed based on the predicted amino acid sequence of their StxA subunits; the remaining four strains contained Stx2c A subunit. Five of the six strains which contained stx(2d-activatable) displayed the activatable phenotype on Vero cells. Genes for adhesins such as the outer membrane protein intimin (eae), which is essential for the intimate attachment and the formation of attaching-and-effacing lesions on intestinal epithelial cells, or the STEC autoagglutinating adhesin (saa), potentially important in eae-negative STEC, were not detected. Moreover, all the strains tested negative for EHEC-hlyA encoding enterohaemorrhagic E. coli (EHEC) hemolysin. To our knowledge, this is the first study that reports the presence of STEC harbouring stx(2d-activatable) and producing the activatable Stx2d in fecal samples of sheep. Therefore both cattle and sheep are reservoirs of such strains and potential sources of human infections. This is of particular importance, because in contrast to other eae-negative STEC, strains producing Stx2d(activatable) may cause severe diseases such as bloody diarrhoea and haemolytic uremic syndrome in humans.     

Escherichia coli 'O' group serological responses and clinical correlations in epidemic HUS patients

This first comprehensive serological analysis of an haemolytic uraemic syndrome (HUS) outbreak in which a wide range of 'O' group Escherichia coli antibody responses in patients and controls provided a unique insight into the epidemiology of such epidemics. Possible answers to clinical aspects related to severity of disease and complications were revealed. A microagglutination assay was used to examine E. coli 'O' group serological responses in 49 serum samples of 21 children hospitalised with HUS and 14 single samples from contemporaneous age-matched controls. A total of 51 O serogroup strains were used, including those reported to be associated with cases of HUS, with six isolates from patients associated with the Adelaide outbreak, environmental verocytotoxi-genic/shiga-toxin producing E. coli (VTEC/STEC) strains and common human commensal strains. Amongst the 21 patients, there were 226 instances of seroreactivity (titre > or = 100) against 34 E. coli serogroups while six instances of seroreactivity against four serogroups occurred in controls. There were 128 instances in patients and one instance in controls in which titres > or = 400 were observed. All 21 patients were seroreactive (titre > or = 100 and <400) to one or more of the 17 HUS-associated serogroups included in the study. Titres ranged from 100 to 6,400, some of the highest in three patients were against O157, whose faeces yielded only EHEC O111, and only one developed O111 antibody. Mixed infection was demonstrated serologically by microagglutination (confirmed by western blot) and was consistent with the multiple serogroups of VTEC found in the mettwurst incriminated as the source, and suggests further strains (not found in the source or in patients' faeces) were probably involved. In HUS-associated EHEC infection, multiple strain infection may be the rule rather than the exception. Analysis of 34 of the 51 serogroup antibody responses in the HUS patients revealed clues to possible relationships with clinical severity and complications. Patients with severe renal failure tended to develop antibodies to a larger number of serogroups than those with moderate or mild impairment. The same was true for central nervous system complications. Other associations were observed. While VTEC O157 remains an important causal serogroup in HUS, non-O157 serogroups also appear to play a significant role and therefore the latter should always be sought in all future HUS cases as new insights into pathogenicity may be discovered. This study indicates that co-infection with different VTEC serogroups may affect clinical outcome.     

Characterisation and clonal relationships of Shiga-toxigenic Escherichia coli (STEC) isolated from Australian dairy cattle

A total of 136 Shiga toxin-producing Escherichia coli (STEC) isolated during a longitudinal survey of three Australian dairy farms were examined to determine their virulence factors, serotype and genomic relationships. This study aimed to assess the potential of these STEC to cause disease in humans and to analyse the on-farm ecology of STEC. Virulence factors (stx, eae, ehxA) were used as determinants of potential to be enterohaemorrhagic E. coli (EHEC) and were examined using polymerase chain reaction (PCR). Among the cattle groups tested, calves, both before and during weaning, shed the most putative EHEC and were the main source of serotypes commonly associated with human disease. E. coli O157:H7 and E. coli O26:H11 represented 9.4 and 7.8% of cattle STEC isolates respectively, with other putative EHEC serotypes reported for the first time from cattle. Based on serotype and virulence factors, 20% of STEC were putative EHEC. Pulsed-field gel electrophoresis (PFGE) was used to compare the genomic profiles of STEC from dairy farms. Isolates common to cattle and the farm environment were identified. Multiple strains of STEC with high clonal turnover were detected in the faeces of cattle, and isolates appeared to be specific to individual farms. To fully assess the pre-slaughter EHEC risk factors on-farm, examination of STEC virulence is as important as determination of STEC prevalence.     

Virulence and fitness gene patterns of Shiga toxin-encoding Escherichia coli isolated from pigs with edema disease or diarrhea in Germany

Fecal Escherichia coli isolates (n = 3,218) from piglets with edema disease or diarrhea were screened for the genes of Stx2 and Stx2 variants. A total of 283 E. coli isolates (8.8%) proved exclusively positive for Stx2e and most of these (85.1%) harbored genes for F18 fimbria. No recognized adhesins were detectable in 14.5% of the isolates. Genes for heat-stable or heat-labile E. coli enterotoxins were found in F18+ as well as F18 isolates (51.9% and 33.3%, respectively). Five isolates also harbored fyuA and irp2 genes which are indicative of a high pathogenicity island in E. coli. All Stx2e+ isolates lacked genes for intimin, EHEC hemolysin, STEC autoagglutinating adhesin, subtilase cytotoxin, serine protease Espl. The majority of Stx2e+ isolates belonged to phylogenetic groups A (59.3%) and D (38.9%) and only few isolates were classified as B1 and B2 (1.8%). The results suggest that Stx2e-producing E. coli strains are highly prevalent in diseased pigs in Germany. Despite their significant diversity, most strains possess all typical features (Stx2e, F18) of porcine edema disease E. coli. However, a considerable portion of porcine strains resembles published human Stx2e+ strains in that they lack any recognized pig-associated adhesin. Thus, a zoonotic potential cannot be excluded for these strains.     

Virulence profiles of Shiga toxin 2e-producing Escherichia coli isolated from healthy pig at slaughter

Recently, virulence patterns of Stx2e-producing Escherichia coli from pigs with edema disease and from humans were compared and strains from diseased pigs were reported to be unlikely human pathogens [Sonntag, A.K., Bielaszewska, M., Mellmann, A., Dierksen, N., Schierack, P., Wieler, L.H., Schmidt, M.A., Karch, H., 2005. Shiga toxin 2e-producing Escherichia coli isolates from humans and pigs differ in their virulence profiles and interactions with intestinal epithelial cells. Appl. Environ. Microbiol. 71, 8855-8863]. In the present study, 31 Shiga toxin-producing E. coli (STEC) strains harboring stx2e, which were previously isolated out of fecal samples from healthy pigs at slaughter [Kaufmann, M., Zweifel, C., Blanco, M., Blanco, J.E., Blanco, J., Beutin, L., Stephan, R., 2006. Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland. J. Food Prot. 69, 260-266], were characterized by phenotypic and genotypic traits. Nine of the thirty-one sorbitol-positive non-O157 STEC (stx2e) isolated from healthy pigs belonged to serotypes found in STEC isolated from humans, including two serotypes (O9:H-, O26:H-) reported in association with hemolytic-uremic syndrome. Otherwise, the serotypes were different from those isolated from cases of edema disease in pigs. The eae (intimin) gene, which is strongly correlated with severe human disease, was not detected. Moreover, all strains were lacking the genes for enterohemolysin (ehxA), porcine A/E associated protein (paa), STEC autoagglutinating adhesin (saa) and the serin protease EspI (espI). Nine strains tested positive for astA (EAST1), one O141:H17 strain for fedA (F18 fimbrial adhesin) and one O159:H- strain for terF (tellurite resistance). Similar to the Stx2e-producing E. coli isolated from humans, which are mainly lacking further virulence factors, genes of an iron uptake system on the high-pathogenicity island (irp2, fyuA) were detected in three ONT:H10 and ONT:H19 strains from healthy pigs. Consequently, although the isolated strains are unlikely to be associated with severe human diseases, healthy pigs cannot be excluded as a potential source of human infection with Stx2e-producing STEC.     

Occurrence and characterization of verocytotoxigenic Escherichia coli (VTEC) strains from dairy farms in Trinidad

A cross-sectional study was conducted to determine the prevalence and characteristics of verocytotoxigenic Escherichia coli (VTEC) on 25 dairy farms each located in Waller field and Carlsen field farming areas in Trinidad. On each selected farm, faecal samples were collected from milking cows, calves and humans; rectal swabs were obtained from pet farm dogs; bulk milk was sampled as well as effluent from the milking parlour. Escherichia coli was isolated from all sources on selective media using standard methods. Isolates of E. coli were subjected to slide agglutination test using E. coli O157 antiserum, vero cell cytotoxicity assay to detect verocytotoxin (VT) and heat labile toxin (LT) production, the polymerase chain reaction (PCR) to detect VT genes, and the dry spot test to screen for E. coli O157 and non-O157 strains. In addition, faecal samples from animal and human sources were tested for VT genes using PCR. Of a total of 933 E. coli isolates tested by the slide test, eight (0.9%) were positive for the O157 strain. The vero cell cytotoxicity assay detected VT-producing strains of E. coli in 16.6%, 14.6%, 3.2% and 7.1% of isolates from cows, calves, farm dogs and humans respectively (P < 0.05; chi(2)). For LT production, the highest frequency was detected amongst isolates of E. coli from calves (10.8%) and the lowest (0.0%) amongst isolates from humans and bulk milk (P < 0.05; chi(2)). Of the 61 VT-producing isolates by vero cell cytotoxicity assay tested by PCR, the VT, LT and eae genes were detected in 62.3%, 4.9% and 1.6% respectively (P < 0.05; chi(2)). Amongst the 45 E. coli isolates that were VT positive (vero cell) or VT-gene positive by PCR, 2.2%, 2.2%, 4.4% and 6.7% belonged to non-O157 strains O91, O111, O103 and O157, respectively, as determined by the Dry spot test. Detection of VTEC strains in milk and dairy animals poses a health risk to consumers of milk originating from these farms. In addition, the demonstration of VTEC strains in humans, VT gene in faecal samples and E. coli isolates as well as non-O157 VTEC strains of E. coli are being documented for the first time in the country.     

Occurrence of enterohaemorrhagic E. coli (EHEC) in domestic animals

Among the verocytotoxin producing E. coli strains (VTEC) the enterohemorrhagic group (EHEC) have emerged as important source of serious disease in human, e.g. the haemolytic uremic syndrome (HUS). VTEC strains possess different virulence profiles where by virulence traits can be provided by the chromosome, by plasmids and, in the case of verocytotoxins (except: VT2e) by bacteriophages. The original and main reservoir are ruminants. In Germany, VTEC strains were isolated in ruminant stocks regularly. In part, the prevalence was estimated up to 100%. However, strains of important EHEC serovar groups, e.g. O157, O26, O111, O103 and O145 as main source of human infections are isolated rarly. This is even the case for food originated from those animals. The hygienic management to avoid fecal contamination of carcasses during the slaughter process is of crucial importance. Future preventive strategies in the field of primary production may be the development of vaccination programs and/or the feeding management to reduce the shedding of acid resistant VTEC. Slowly recognized environmental sources of infection and contamination are biotic (e.g. flys, rodents) and abiotic factors (e.g. pasture, water, feed). In an own study that investigated the prevalence of VTEC positive animals in free range cows during sojourn on pasture a significant increase was estimated. Even asymptomatic human carriers can serve as source of infection or contamination.