Environmental air pollution and DNA adducts in Copenhagen bus drivers--effect of GSTM1 and NAT2 genotypes on adduct levels

Fecal samples from 335 dairy farm residents and 1458 cattle on 80 farms were tested for Vero cytotoxin (VT)-producing Escherichia coli (VTEC). Residents were also tested for antibodies to VT1 and O157 lipopolysaccharide (LPS). Residents and cattle on farms with VTEC-positive persons or E. coli O157:H7-positive cattle were retested. Twenty-one persons (6.3%) on 16 farms (20.8%) and 46% of cattle on 100% of the farms had VTEC in fecal samples. Human VTEC isolates included E. coli O157:H7 and 8 other serotypes, 4 of which were present in cattle on the same farms. More persons had antibodies to VT1 (41%) than to O157 LPS (12.5%). Seropositivity to O157 LPS was associated with isolation of E. coli O157:H7 on the farm (P = .022). Human VTEC infection was negatively associated with age (P < .05) and was not associated with clinical illness. Many dairy farm residents experience subclinical immunizing VTEC infections at a young age, which frequently involve non-O157 VTEC found in cattle.     

The use of serodiagnosis in the retrospective investigation of a nursery outbreak associated with Escherichia coli O157:H7

AIMS: To use serology to investigate an outbreak of verocytotoxin (VT) producing Escherichia coli O157 in a hospital nursery, following the detection of faecal E coli O157 (phage type 49) producing VT type 2. METHODS: ELISA and immunoblotting techniques, based on lipopolysaccharide (LPS) purified from E coli O157; diagnostic bacteriology; serotyping and phage typing; DNA probes for VT. RESULTS: 29 of 126 sera contained antibodies to the LPS of E coli O157: 10 were from children, three were from staff, and 11 were from hospital kitchen staff. Five parents of children attending the nursery were antibody positive. Sixty four sera from other hospital staff and controls did not contain antibodies to the LPS of E coli O157. CONCLUSIONS: Serology detected evidence of infection with E coli O157 in 23% of sera examined. By bacteriology alone, only a single case of infection with E coli O157 would have been detected. Serology is valuable in providing evidence of infection with E coli O157.     

Occurrence of verocytotoxin-producing Escherichia coli O157 on Dutch dairy farms

During the period from September 1996 through November 1996, 10 Dutch dairy farms were visited to collect fecal samples from all cattle present. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. Cattle on 7 of the 10 dairy farms tested positive for O157 VTEC, with the proportion of cattle infected varying from 0.8 to 22.4%. On the seven farms positive for O157 VTEC, the excretion rate was highest in calves ages 4 to 12 months (21.2%). In a follow-up study, two O157 VTEC-positive farms and two O157 VTEC-negative farms identified in the prevalence study were revisited five times at intervals of approximately 3 months. Cattle on each farm tested positive at least once. The proportion of cattle infected varied from 0 to 61.0%. Excretion rates peaked in summer and were lowest in winter. Again, the highest prevalence was observed in calves ages 4 to 12 months (11.8%). O157 VTEC strains were also isolated from fecal samples from horses, ponies, and sheep and from milk filters and stable flies. O157 VTEC isolates were characterized by VT production and type, the presence of the E. coli attaching-and-effacing gene, phage type, and pulsed-field gel electrophoretic genotype. No overlapping strain types were identified among isolates from different farms except one. The predominance of a single type at each sampling suggests that horizontal transmission is an important factor in dissemination of O157 VTEC within a farm. The presence of more than one strain type, both simultaneously and over time, suggests that there was more than one source of O157 VTEC on the farms. Furthermore, this study demonstrated that the O157 VTEC status of a farm cannot be ascertained from a single visit testing a small number of cattle.     

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An analysis of farm workers and rural dwellers for serum antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157 detected sera with antibodies binding to bovine serum albumin (BSA) by ELISA. These antibodies were not specific for BSA when examined by immunoblotting, and the ELISA values were reduced to a background level when plates were blocked with normal rabbit serum.     

Interferon-induced protein 10 and interleukin 8. C-X-C chemokines present in proliferative diabetic retinopathy

From May through November 1997, 1,011 samples of raw milk from bulk storage tanks were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. The samples originated from 1,011 different dairy herds located throughout the Netherlands. O157 VTEC was not isolated from any of the milk samples examined. Additionally, survival of O157 VTEC in raw and UHT-sterilized cow's milk at 7 and 15 degrees C was studied, both in the absence and presence of an activated lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS). Results indicated that the O157 VTEC strain tested was able to grow in raw milk at 7 degrees C as well as at 15 degrees C. Naturally occurring amounts of thiocyanate and hydrogen peroxide in the raw milk tested were not sufficient to activate the LPS. Although the LPS exhibited an antimicrobial activity against O157 VTEC in LPS-activated sterilized milk, O157 VTEC populations were not (or not as obviously) reduced in LPS-activated raw milk. Possibly background microflora were more sensitive to the LPS than the O157 VTEC test strain. It was concluded that raw milk contaminated with O157 VTEC will remain a hazard if kept at 7 or 15 degrees C. Effective pasteurization and avoiding postpasteurization contamination are necessary to ensure the safety of milk.     

Serological detection of verocytotoxin-producing Escherichia coli in patients with haemolytic uraemic syndrome in western Europe

Sera from 45 patients from The Netherlands, Germany and Belgium who had a clinical diagnosis of haemolytic uraemic syndrome (HUS) were screened for antibodies to the lipopolysaccharide (LPS) of Escherichia coli producing Verocytotoxin (VTEC). Sera from 43 family contacts and 34 control patients were also examined. Using the techniques of EIA and immunoblotting, antibodies to the LPS of Escherichia coli O157 were found in 28 patients, and to the LPS of serogroups O115 and O145 in one patient and one family member respectively. The results of our study suggest that VTEC, and in particular Escherichia coli O157, might be a significant cause of HUS in Western Europe.     

Isolation and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep

In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup 0157. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of E. coli O157 strains that are potentially pathogenic for humans.     

Identification of new verocytotoxin type 2 variant B-subunit genes in human and animal Escherichia coli isolates

The sequence of a verocytotoxin 2 (VT2) variant gene that was untypeable by the B subunit PCR and restriction fragment length polymorphism analysis (PCR-RFLP) method described by Tyler et al. (S. D. Tyler, W. M. Johnson, H. Lior, G. Wang, and K. R. Rozee, J. Clin. Microbiol. 29:1339-1343, 1991) was determined and compared with published sequences. It was highly homologous to two recently reported VT2 variant sequences. The PCR-RFLP method described by Tyler et al. was extended to include these new sequences. New VT2 variants were identified in 65 of 359 VT-producing Escherichia coli (VTEC) with newly designed primers (VT2-cm and VT2-f) and were characterized as well by restriction analysis of the amplification products obtained with another VT2-specific primer pair (VT2-e and VT2-f). The VT genes harbored by 64 of these isolates proved to be untypeable by Tyler's PCR-RFLP method because no amplification was obtained with the primers used with this method (VT2-c and VT2-d). The last isolate harbored the new variant gene in addition to VT2vh-a. None of the isolates harboring these new toxin genes belonged to serogroups O157, O26, O103, O111, and O145. All 65 isolates were negative for the eaeA gene and were significantly less frequently enterohemolytic or positive for the enterohemorrhagic E. coli (EHEC) virulence plasmid than non-O157 VTEC isolates harboring other VT2 genes. They were also less frequently isolated from patients with EHEC-associated symptoms. The extended PCR-RFLP typing method is a useful tool to identify less-virulent VTEC isolates and for VT genotyping in epidemiological studies with non-O157 strains.     

Verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC in 1-30-days-old diarrhoeic dairy calves

Faecal samples from 221, 1-30-days-old, diarrhoeic dairy calves were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC. Calves were grouped according to their age (1-7, 8-14, 15-21 and 22-30 days) and analyses of prevalences were done by Mantel-Haenzsel chi 2-test for trend. VTEC and eae-positive non-VTEC were detected in 20 (9.0%) and 18 (8.1%) of the diarrhoeic calves, respectively. A significant age-associated increase in the prevalence of VTEC (p = 0.0001), but not in the prevalence of eae-positive non-VTEC (p = 0.381), was found. Significant differences in VTEC prevalence were found between the age-group 22-30 days and in all other age-groups. 43 (5.0%) of the 861 E. coli isolates from the 221 diarrhoeic calves were VTEC, and 30 (69.8%) of these strains produced VT1 only. More than one-half of the VTEC strains (55.8%) were positive for the eae gene and all these eae-positive VTEC strains produced VT1 only. A high percentage (76.7%) of VTEC strains belonged to E. coli serogroups (O4, O26, O39, O91, O113, O128 and O145) associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. 51 (5.9%) of the E. coli strains studied were eae-positive non-VTEC and the serogroups most prevalent among these strains were O4, O14, O26 and O123. Only four of the eae-positive strains were also espB-positive by hybridization with a probe from a human EPEC isolate and none of these strains produced VT.     

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A cluster of four cases of haemolytic uraemic syndrome in children occurred in Northern Bohemia, Czech Republic, between 15 June and 7 July, 1995. All the cases had significantly elevated titres of anti-O157 lipopolysaccharide (LPS) antibodies as detected by the indirect haemagglutination assay. All but one of them had drunk unpasteurized goat's milk from the same farm within the week before the disease. Evidence of E. coli O157 infection was subsequently found in 5 of 15 regular drinkers of the farm's raw goat's milk; four of them were asymptomatic, 1 had mild diarrhoea at the end of June. Verocytotoxin 2-producing E. coli O157:H7 strains of phage type 2 and of identical pulsed-field gel electrophoresis patterns were isolated from 1 of 2 farm goats and from 1 of the asymptomatic goat's milk drinkers. The frequency of anti-O157 LPS antibodies found among regular drinkers of the farm's raw goat's milk (33%; 5 of 15) was significantly higher than that found in control population (0%; none of 45) (P = 0.0005; Fisher's exact test). Our findings indicate that goats may be a reservoir of E. coli O157:H7 and a source of the infection for humans; raw goat's milk may serve as a vehicle of the pathogen transmission.     

Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin

A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.     

Enterohaemorrhagic Escherichia coli O157:H7 infection

Escherichia coli O157:H7 differs from previously described diarrheagenic E. coli classes (enteropathogenic, enteroinvasive, enterotoxigenic) by distinct clinical symptoms, production of verotoxin (VT) and a specific plasmid. Cattle are the primary reservoirs of E. coli O157:H7. The organism may be transmitted through the consumption of contaminated foods (mainly of bovine origin) and by person-to-person contact. The most typical clinical manifestations of E. coli O157:H7 infection are hemorrhagic colitis and hemolytic-uremic syndrome. Since the 1982 many outbreaks of E. coli O157:H7 infections as well as sporadic cases have been documented. Diagnosis of E. coli O157:H7 is based on a positive stool culture, presence of VT and elevated serum antibodies. The best currently available and inexpensive method for diagnosing E. coli O157:H7 is culture of stool on sorbitol-Mac Conkey agar medium.     

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

A study for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E. coli) O157 by the bacterial agglutination technique

We evaluated the usefulness of bacterial agglutination antibodies for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E. coli) O157 infections. We examined 50 serum samples from 50 control children (whiout diarrhea 31, with diarrhea 19), 24 samples from 8 diarrhea cases due to O157:H7, 37 samples from 14 cases of hemolytic uremic syndrome (HUS) for antibodies to heat-killed E. coli E32511 (O157:H.-) strain using the bacterial agglutination technique. Of the control sera all but one (x80) showed 20 > or = in the antibody. All the diarrhea patients due to O157:H7 showed a significant rise (x160-x5120) of the titers in the sera at 5-7 days on illness, after that the titers fell rapidly. Significant antibody rise (x160-x5120) was detected in twelve out of 14 HUS patients at the early stage of the illness which fell in the convalescent phase. The assay appeared to be a useful serodiagnostic technique because of its easiness and simplicity as well as because of its high sensitivity and specificity.     

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA): optimal conditions for quantitation of antibodies

In the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) the quantitation of antibodies is based on their ability to form a diffusion gradient over an antigen-coated polystyrene surface. The antigen-antibody reaction is then visualized by an enzyme-conjugated anti-immunoglobulin. The enzyme-substrate reaction is finally performed by pouring a substrate-containing gel over the polystyrene surface. In this study with bovine serum albumin as antigen and a corresponding rabbit antiserum, the diffusion time of antiserum was shown to be the most critical variable of the method, while the antigen concentration used for coating, the conjugate binding time and the enzyme-substrate reaction time had a minor influence on the quantitation of antibodies. High antibody levels were measured with greater accuracy than low levels, but the standard deviation was below 10%. It was also shown that different sera containing antibodies to Salmonella typhi O LPS, Klebsiella pneumoniae K1 and O4 LPS, Escherichia coli O2 LPS, Yersinia enterocolitica Y3 LPS, cardiolipin and pneumococcus could be quantitated with the same accuracy.     

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Management factors in 36 Pacific Northwest dairy herds were evaluated for their association with the prevalence of Shiga toxin-positive Escherichia coli O157 (E. coli O157) in dairy cattle. The within-herd prevalence of E. coli O157 was estimated by bacteriological culture of fecal pat samples, collected monthly for 6 months (approximately 60 per visit), from heifer cattle. During the first visit to each farm, a management questionnaire was administered that covered a broad range of animal husbandry practices. On each subsequent visit, a brief questionnaire was administered to detect changes in management practices. A significantly higher prevalence of E. coli O157 was noted in herds that fed corn silage to heifers compared to herds that did not feed corn silage. More tentative associations of E. coli O157 prevalence were observed for weaning method, protein level of calf starter, feeding of ionophores in heifer rations, feeding of grain screens to heifers, and feeding of animal by-products to cows.     

Opsonization of Actinobacillus actinomycetemcomitans by immunoglobulin G antibodies to the O polysaccharide of lipopolysaccharide

Sera of localized juvenile periodontitis (LJP) patients colonized by Actinobacillus actinomycetemcomitans serotype b often contain markedly elevated levels of immunoglobulin G (IgG) antibodies to serospecific determinants in the O polysaccharide of lipopolysaccharide (LPS), as well as to outer membrane proteins of this species. IgG antibodies in LJP sera are known to opsonize A. actinomycetemcomitans for subsequent phagocytosis and killing by human neutrophils. The objective of this study was to determine whether outer membrane proteins or serospecific determinants in LPS are the primary target for opsonic IgG antibodies in LJP sera. An A. actinomycetemcomitans serotype b O-polysaccharide affinity column was constructed and subsequently used to purify LPS-specific IgG antibodies from LJP serum. The affinity-purified anti-LPS IgG antibodies were enriched in content of IgG2 (66.2%, compared with 37.0% in the total IgG fraction) and were immunospecific for A. actinomycetemcomitans serotype b LPS. In an opsonophagocytic assay using neutrophils from donors who were homozygous for the H131 allotype of Fcy receptor IIa (CD32), it was found that LPS-specific IgG antibodies exhibited substantially greater opsonic activity toward A. actinomycetemcomitans serotype b than an LJP IgG fraction that was depleted of LPS-reactive antibodies but contained antibodies against outer membrane proteins of this species. The results of this study indicate that serospecific determinants in the O polysaccharide of A. actinomycetemcomitans serotype b are a principal target for opsonic antibodies in sera of LJP subjects.     

Increased immunoglobulin binding to cerebral endothelium in patients with antiphospholipid antibodies

BACKGROUND AND PURPOSE: There is a strong link between antiphospholipid antibodies and stroke. The mechanism of action of antiphospholipid antibodies is unknown. Most theories of pathogenesis center around platelet or endothelial cell dysfunction. Our aim was to determine if there were immunoglobulins in the sera of patients with antiphospholipid antibodies that bind human brain microvascular endothelial cells. METHODS: We studied sera from three groups of subjects: patients with antiphospholipid antibodies and stroke (group 1), healthy control subjects (group 2), and patients with stroke but without antiphospholipid antibodies (group 3). We isolated human brain microvascular endothelial cells from temporal lobectomy specimens and used a cellular enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin binding to endothelial cells derived from human brain and from human umbilical vein. We used a chromium release assay to measure cytotoxicity. RESULTS: Patients with antiphospholipid antibodies and stroke had significantly higher immunoglobulin binding to human brain microvascular endothelial cells than subjects in the other groups ([ELISA index+standard deviation], 63 +/- 37 [group 1] versus 7 +/- 7 [group 2] versus 7 +/- 7 [group 3], P < .001). There was, however, poor correlation between binding to brain endothelial cells and binding to cardiolipin. The binding to brain microvascular cells was not specific to brain endothelium, as similar results were found in an ELISA using human umbilical vein cells. There was no evidence of complement-mediated brain endothelial cell cytotoxicity. CONCLUSIONS: Patients with stroke and antiphospholipid antibodies frequently have human brain microvascular endothelial-reactive antibodies in their serum. These antibodies are distinct from those to cardiolipin. We found no evidence that these antibodies are cytotoxic.     

Studies on enrichment broth for verotoxin-producing Escherichia coli (VTEC) O157

Growth of verotoxin-producing Escherichia coli (VTEC) O157 in conventionally recommended pre-enrichment broth media at different temperatures was evaluated. In addition, sensitivity of VTEC O157 isolates to several antibacterial drugs, which were added to the selective enrichment broth, was tested. All five isolates of VTEC O157 tested grew well in trypticase soy broth (TSB) at 36 degrees C and 42 degrees C, while the growth of one isolate was markedly suppressed in TSB supplemented with cefixime (CFIX), potassium tellurite (PT), and vancomycin (TSB-CTV) even at 36 degrees C. A significant growth suppression was also observed in three of the isolates cultured in novobiocin (NB)-supplemented modified EC broth (mEC-NB) at 42 degrees C. In mEC-NB after 24-hr incubation at 36 degrees C, the five VTEC O157 isolates grew well, although one isolate was slightly suppressed during the first 8 hours. Minimum growth inhibitory concentrations of CFIX, NB and PT for a total of 90 clinical and environmental isolates of VTEC O157 were all above the concentrations usually prescribed for mEC-NB and TSB-CTV. These findings suggest that mEC-NB and TSB-CTV should be used at 36 degrees C for growth of VTEC O157 and that use of a nonselective pre-enrichment broth medium (i.e. TSB) together with a selective one (i.e. TSB-CTV or mEC-NB) is necessary for successful isolation of VTEC O157 from various specimens.     

Over-expression of acetylcholinesterase stimulates the expression of agrin in NG108-15 cells

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.