Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand.

Specific DNA probes were used to identify Shiga-like toxin I (SLT I)- and SLT II-producing Escherichia coli in vegetables, meats, cattle, and farm animals in Thailand. SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E. coli types that hybridized with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E. coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the SLT II probe, and 44% hybridized with both SLT probes. The enterohemorrhagic E. coli plasmid probe hybridized with 64% (68 of 106) of SLT-producing E. coli isolates from food and cattle and with 8% (17 of 201) of E. coli isolates from pigs. No SLT-producing E. coli was detected in pigs. Seventy-six percent (26 of 34) of E. coli isolates that hybridized with the SLT II probe were cytotoxic to Vero but not to HeLa cells, suggesting that they produced the variant of SLT II. The high prevalence of SLT-producing E. coli in beef-producing animals suggests that exposure to animals and eating beef may pose a health risk for acquiring enterohemorrhagic E. coli infections in Thailand.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Identification of uidA gene sequences in beta-D-glucuronidase-negative Escherichia coli

A probe specific for the uidA gene of Escherichia coli hybridized with 112 of 116 E. coli isolates examined, including 31 beta-D-glucuronidase-negative and 12 enterohemorrhagic E. coli serotype O157:H7 isolates. Southern hybridizations confirmed the presence of a 900-bp HinfI fragment from the uidA gene in all isolates examined, suggesting that uidA gene sequences are present in most E. coli.     

Identification of uidA gene sequences in beta-D-glucuronidase-negative Escherichia coli.

A probe specific for the uidA gene of Escherichia coli hybridized with 112 of 116 E. coli isolates examined, including 31 beta-D-glucuronidase-negative and 12 enterohemorrhagic E. coli serotype O157:H7 isolates. Southern hybridizations confirmed the presence of a 900-bp HinfI fragment from the uidA gene in all isolates examined, suggesting that uidA gene sequences are present in most E. coli.     

Growth of Escherichia coli in the presence of trimethoprim-sulfamethoxazole facilitates detection of Shiga-like toxin producing strains by colony blot assay

Abstract Subinhibitory concentrations of trimethoprim-sulfamethoxazole increased the total yield of Shiga-like toxin (SLT), produced by Shigella dysenteria 1 and by enterophathogenic and enterohemorrhagic strains of Escherichia coli . Stimulation of SLT synthesis by trimethoprim-sulfamethoxazole was demonstrated by an increase in cytotoxic activity for HeLa cells and the diameter of the zone formed around bacterial colonies probed with monoclonal antibodies to SLT. Thus, supplementation of culture media with trimetroprimsulfamethoxazol will facilitate SLT purification and detection of SLT-producing bacteria.     

Synthetic oligodeoxyribonucleotide probes to detect verocytotoxin-producing Escherichia coli in diseased pigs

Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E. coli O138 and O139 strains. By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp. By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E. coli producing SLT-IIp and E. coli strains associated with human disease producing SLT-II. Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II. The toxins were distinguishable in their ability to kill HeLa S-3 cells. The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E. coli to further clarify their role in diseased pigs.     

Evaluation of DNA probes for detection of Shiga-like-toxin-producing Escherichia coli in food and calf fecal samples.

The use of DNA probes for Shiga-like toxin I (SLT-I) and SLT-II for detection of SLT-producing Escherichia coli (SLTEC) in foods and calf fecal samples was evaluated. Enrichment cultures were prepared from food or fecal samples. Colonies formed by plating of enrichment cultures were probed for SLTEC by colony hybridization. Alternatively, enrichment cultures were analyzed for SLTEC presence by dot blot. The lowest detected concentration of SLTEC in sample homogenates inoculated with E. coli O157:H7 corresponded to 1.3 CFU/g of sample. Of the 44 food samples and 28 fecal samples from dairy calves tested by the colony hybridization method, 4 food samples, including ground beef, raw goat milk, blueberries, and surimi-based delicatessen salad, and 9 calf fecal samples were positive with the SLT probes. The dot blot technique yielded results within 48 h and can be used as a fast and sensitive method of detection for SLTEC in foods and calf fecal samples. The colony hybridization technique took 3 to 4 days but permits recovery of the positive colonies when desired.     

Extraction of lithium from spodumene by bioleaching

A multiplex PCR assay specifically detecting Escherichia coli O157 : H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157 : H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157 : H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55 : H7 and E. coli 055 : NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.     

Evaluation of DNA probes for detection of Shiga-like-toxin-producing Escherichia coli in food and calf fecal samples

The use of DNA probes for Shiga-like toxin I (SLT-I) and SLT-II for detection of SLT-producing Escherichia coli (SLTEC) in foods and calf fecal samples was evaluated. Enrichment cultures were prepared from food or fecal samples. Colonies formed by plating of enrichment cultures were probed for SLTEC by colony hybridization. Alternatively, enrichment cultures were analyzed for SLTEC presence by dot blot. The lowest detected concentration of SLTEC in sample homogenates inoculated with E. coli O157:H7 corresponded to 1.3 CFU/g of sample. Of the 44 food samples and 28 fecal samples from dairy calves tested by the colony hybridization method, 4 food samples, including ground beef, raw goat milk, blueberries, and surimi-based delicatessen salad, and 9 calf fecal samples were positive with the SLT probes. The dot blot technique yielded results within 48 h and can be used as a fast and sensitive method of detection for SLTEC in foods and calf fecal samples. The colony hybridization technique took 3 to 4 days but permits recovery of the positive colonies when desired.     

Detection and identification of pathotypes of verocytotoxigenic Escherichia coli isolated from weaned piglets using gene probes for seven E. coli toxins

Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII). The results with the EDP and SLTII probes were identical. All VTEC hybridized with these two probes, while non VTEC did not. All 133 E. coli were negative for the SLTI probe. Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP. The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins. Only the STb probe was hybridized by 36 of them. Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC.     

Genetic diversity of Escherichia coli recovered from the oral cavity of beef cattle and their relatedness to faecal E. coli

AIMS: To determine the genetic diversity of generic Escherichia coli recovered from the oral cavities of beef cattle and their relatedness to E. coli isolated from the faeces of cattle during pasture grazing and feedlot finishing. METHODS AND RESULTS: A total of 484 E. coli (248 oral and 236 faecal isolates) were obtained from eight beef cattle after 1 and 5 months of grazing on pasture and after 1 and 5 months in a feedlot. The random amplification of polymorphic DNA (RAPD) method was used to genetically characterize these isolates. The RAPD patterns showed that ca 60% of E. coli recovered from the oral cavities and faeces during pasture and feedlot shared a close genetic relatedness. A number of E. coli with unique RAPD types were also found either in the oral cavities or faeces. Most of the E. coli RAPD types recovered from the oral cavities were shared among animals, but there were also RAPD types which were unique to individual animals. The E. coli populations of the oral cavities were genetically diverse and changed over time. CONCLUSIONS: This study indicates that there are large numbers of E. coli carried in the oral cavities of beef cattle and those E. coli are closely related to strains found in the faeces. The oral cavities of cattle harbour a genetically diverse E. coli population. SIGNIFICANCE AND IMPACT OF THE STUDY: The oral cavity may be an important reservoir of enteric pathogens which may transfer to meat during carcass dressing. A better understanding of the molecular ecology of E. coli in cattle would assist the design of approaches to control pathogenic strains during beef production and processing.     

Prevalence of enterohemorrhagic Escherichia coli in raw and treated municipal sewage.

Municipal sewage was screened for DNA encoding Shiga-like Toxin (SLT) II, a key protein involved in the virulence of enterohemorrhagic Escherichia coli. PCR analysis of sewage concentrates showed that DNA encoding SLT II was present in a single sample of untreated sewage and absent in all other samples tested (n = 6). Thermotolerant E. coli cultured from the sewage (n = 1,520) also tested negative for SLT II by colony hybridization.     

Origin of contamination and genetic diversity of Escherichia coli in beef cattle

The possible origin of beef contamination and genetic diversity of Escherichia coli populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene. E. coli was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter. The 1,403 E. coli isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method. Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef. E. coli genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals. The genetic diversity of the E. coli population changed over time within individual animals grazing on pasture and in the feedlot. Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the fliC gene and were grouped into eight genotypes. There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques. In conclusion, the E. coli contaminating meat can originate from cattle feces, and the E. coli population in beef cattle was highly diverse. Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing.     

DNA polymorphism in the major histocompatibility complex of man and various farm animals*

Summary. In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15–25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificites. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies. This is even more so in large farm animals where for various reasons it is almost impossible to conduct certain types of investigation that are easily performed in rodents or in man. Although the data are still preliminary, they already extend our knowledge of the MHC in domestic animals far beyond what could have been reasonably anticipated using conventional methods.     

Correlation of genes in the pap gene cluster to expression of globoside-specific adhesin by uropathogenic Escherichia coli

Abstract Expression of globoside-specific pilus adhesin of Escherichia coli is the virulence factor most commonly associated with pyelonephritis. In the clinical isolate J96 (O4:K6:H5) expression of globoside binding pili require the proteins encoded by the papE, papF , and papG genes in the pap gene cluster. Probes derived from these genes were used in dot blot hybridization analysis of E. coli urinary tract isolates obtained from patients with significant bacteriuria. Fecal E. coli isolates from healthy individuals were also analyzed. The probe encompassing the papF and papF J96 genes hybridized to all urinary tract infectious (UTI) isolates expressing globoside-specific adhesin, whereas papG J96 only hybridized to the strain from which the fragment was cloned. In contrast, a papG -specific probe from the O:6 strain IA2 hybridized to all but one of the UTI isolates that expressed the adhesin. In both materials, but especially among the fecal isolates, strains were found that hybridized to the probes but did not express the adhesin. The data shows that papEF -specific DNA can be used for the diagnosis of potentially pyelonephritic E. coli .     

DNA polymorphism in the major histocompatibility complex of man and various farm animals

In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15-25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificities. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Occurrence of Shiga-like toxin-producing Escherichia coli in retail fresh seafood, beef, lamb, pork, and poultry from grocery stores in Seattle, Washington.

Fresh meat, poultry, and seafood purchased from Seattle area grocery stores were investigated for the presence of Shiga-like toxin-producing Escherichia coli by using DNA probes for Shiga-like toxin (SLT) genes I and II. Of the 294 food samples tested, 17% had colonies with sequence homology to SLT I and/or SLT II genes.     

Sources of Escherichia coli O157 and experiences over the past 15 years in Sheffield, UK

In the first documented outbreak of HC caused by Escherichia coli O157, which occurred in the North-west USA in 1982, there was a strong association between infection and prior consumption of ground beef from a chain of fast food restaurants. Foods of bovine origin, including beef, milk and dairy products, have since been implicated in many outbreaks of infection world-wide. Investigations during the course of outbreaks, or at random, have shown that cattle are a major reservoir of E. coli O157. E. coli O157 was isolated from cattle at slaughter in Sheffield in 1987, this being the first isolation from cattle in the UK. Following a cluster of cases in May/June 1992, an abattoir study showed the organism to be present in 4% of cattle at slaughter and on up to a third of carcasses from rectal swab-positive animals. E. coli O157 was isolated from a food source (unpasteurized milk), for the first time in the UK, in Sheffield in May 1993. During surveillance in 1995-6, E. coli O157 was isolated from 15.7% of cattle, with a monthly prevalence which varied from 5 to 37%. E. coli O157 was also isolated from 2.2% of sheep. During surveillance in 1996, E. coli O157 was isolated from 5.9% of samples of lamb products and from 1.5% of samples of beef products, despite the prevalence in cattle being much higher than in sheep. Work is in progress to try to explain this higher prevalence in lamb products. During 1997 in Sheffield, the only cases of E. coli O157 for which a confirmed source was established were associated with direct animal contact on farm visits. During on-farm investigations of these cases, E. coli O157 was isolated from faecal samples from adult cattle, calves, three different breeds of sheep, two different breeds of pigs, goats and a pony.     

Intestinal parasite infections in pigs and beef cattle in rural areas of Chungcheongnam-do, Korea

The present study was performed to investigate the infection status of intestinal parasites in pigs and beef cattle in rural areas of Chungcheongnam-do, Korea. From November 2009 to April 2010, a total of 241 fecal samples of pigs and beef cattle (136 and 105, respectively) were examined by direct smear and centrifugal sedimentation methods. The overall positive rates of intestinal parasites among pigs and beef cattle were 73.5% and 4.8%, respectively, and the double-infection rate was 10.3% in pigs. Of 136 specimens from pigs, Balantidium coli, Ascaris suum, and Entamoeba spp. infections were found in 88 (64.7%), 24 (17.6%), and 5 cases (3.7%), respectively. Of 105 beef cattle, Entamoeba spp. infections were detected in 5 cases (4.8%). From these results, it is shown that pigs raised on rural farms in Chungcheongnam-do had a high B. coli infection rate and a moderate A. suum infection rate. These results demonstrate that environmentally resistant cysts or eggs could be widespread on the farms examined, and thus an effective hygienic management system is needed to prevent them from serving as the source of infection for human beings.