Effects of cutting process on pork meat contamination by verotoxin-producing Escherichia coli (VTEC) and E. coli O157:H7.

The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) prevalence in pork cutting meat; (ii) to determine the effects of cutting process on pork meat contamination by VTEC; (iii) to characterise the VTEC strains isolated from pork and pork cutting plants (virulence genes and serotype); and (iv) to compare the strains isolated the same day in the same cutting plant in order to identify the routes of contamination inside the cutting plant. Pork carcasses from three French cutting plants were sampled before carcass cutting (carcass samples), after carcasses were divided into big portions (untrimmed cuts) and after preparation of primal cuts (rindless boneless cuts), and different environmental sites in each cutting plant were sampled at three different times in the work day. Potable water was also collected. PCR detection of stx genes was performed on a total of 2042 samples. In addition, a second PCR specific for E. coli O157:H7 detection was carried out on the stx-positive samples. VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2e, uidA and genes which are associated with virulence) and pulsotyped. No E. coli O157:H7 was detected. Meat contamination decreased from carcass (12%) and primary cuts (19%) to secondary cuts (5%), whereas environmental contamination increased after 2 h of activity (from 3% before the commencement of the work day to 25% and 20%, 2 and 6 h after commencement of cutting). No VTEC isolates harboured eae, ehx and uidA genes. VTEC contamination routes were not clearly identified.

Effects of slaughter processes on pig carcass contamination by verotoxin-producing Escherichia coli and E. coli O157:H7.

The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) faecal carriage of slaughtered pigs; (ii) to determine the effects of three different pig slaughtering processes on pig carcass contamination by VTEC; (iii) to characterise the VTEC strains isolated from pig and pig slaughterhouses (virulence genes and serotype); and (iv) to compare the strains isolated in the same slaughterhouse in order to identify the routes of contamination inside the slaughterhouse. Pork carcasses from three French slaughterhouses were sampled at three steps of the slaughter process and different sites in each slaughterhouse were sampled at three different times in the work day. Faecal material from each sampled carcass, potable water and scalding water were also collected. Detection of stx genes was performed by polymerase chain reaction (PCR) on a total of 1227 samples. In addition, a second PCR specific for E. coli O157:H7 detection was carried out on the stx-positive samples. VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2c, uidA genes associated with virulence) and pulsotyped. No E. coli O157:H7 was isolated from the three uidA-positive samples. VTEC faecal carriage was 31%. Global carcass contamination decreased with slaughter process (from 46% to 15%), whereas environmental contamination increased (from 7% to 29%). No VTEC isolates harboured eae, ehx, and uidA genes. VTEC contamination routes were not clearly identified.

Prevalence of verotoxin-producing Escherichia coli (VTEC) and E. coli O157:H7 in French pork.

AIMS: To determination the prevalence of VTEC in pork products and the surrounding environment of the pork plant (slaughterhouse and cutting plant), and characterization of the VTEC strains isolated (virulence genes and serotype). METHODS AND RESULTS: Among the 2146 carcass and pork samples and 876 environmental samples (swabs of surfaces or materials), 328 (15%) and 170 (19%) were PCR-positive for stx genes respectively. VTEC strains were recovered from positive samples by colony hybridization or immunoconcentration, serotyped and genetically characterized. Strains of E. coli O157:H7 were not isolated from 3 uidA-positive samples detected by PCR. The VTEC isolates did not harbour eae, ehx and uidA genes. CONCLUSIONS: Pigs and pork meat may contain VTEC strains but characterization of the strains based on virulence factors showed that the potential danger of pork meat appears to be low since although all strains harboured a stx gene, they did not have other virulence genes. SIGNIFICANCE OF THE STUDY: General hygiene measures appear to be sufficient and specific hygiene measures for VTEC are not necessary at this time. The porcine VTEC strains isolated in our study probably do not present a hazard.

VIDAS enzyme-linked immunoflourescent assay for detection of Listeria in foods: collaborative study.

The VIDAS LIS method and the traditional culture methods for detection of Listeria species in food were evaluated in a multilaboratory comparative study. The 6 foods tested were either naturally contaminated or inoculated with 3 different concentrations of Listeria. Results for each food and each contamination level with the VIDAS LIS method were as good as or better than those obtained with the traditional culture method. Of 1558 samples tested, 935 were positive: 839 by the VIDAS method and 809 by standard culture methods. Overall false negative rates were 10.3 and 13.5% for the VIDAS LIS and culture methods, respectively. The false positive rate for the VIDAS LIS assay was 1.4% based on 9 VIDAS LIS positive assays that did not confirm positive by isolation of Listeria. The agreement between the VIDAS LIS and culture methods for all samples tested was 86%.

Detection of Escherichia coli O157:H7 in heifers' faecal samples using an automated immunoconcentration system.

Pre-treatment of a 5-h enrichment culture with an automated immunoconcentration (ICE) system greatly improved the isolation of Escherichia coli O157:H7 from spiked heifer faecal samples. Enrichment samples plated directly onto sorbitol MacConkey agar (SMAC) and SMAC agar supplemented with cefixime and potassium tellurite (CT-SMAC) showed recovery rates of 8% and 56%, respectively. However, after ICE treatment, E. coli O157:H7 was recovered from 92% of the samples on SMAC and 100% on CT-SMAC. Immunoconcentration analysis of heifers' faecal samples collected from a slaughter-house in France, during March to June 1998, showed that 1% (three of 300) was positive for E. coli O157:H7. Phenotypic and genotypic analysis showed that all three isolates carried both the O157 and H7 antigens, did not ferment sorbitol or had beta-glucuronidase activity and carried trait virulence factors for E. coli O157:H7 (uidA allele, eaeA and pO157 plasmid). However, only one strain was toxigenic and this strain produced a single toxin, namely verotoxin 2.

An automated method for the detection of staphylococcal heat stable deoxyribonuclease in dairy products.

An automated sandwich immunoassay with specific polyclonal antibodies for the detection of Staphylococcus aureus thermostable nuclease (DNase) is described. To evaluate this assay, different quantities of purified S. aureus nuclease were added to dairy products. Additionally, staphylococcal counts and nuclease activity of milk samples inoculated with S. aureus were determined. Different extraction procedures were performed and compared. The results indicated that the automated test was a reliable method for detecting DNase activity in milk products. The procedure was completed in 2 h and detected 1 ng of DNase ml-1. Detection of the DNase was especially useful in cheeses and could be used to confirm positive enterotoxin results.

Evaluation of an alternative extraction procedure for enterotoxin determination in dairy products.

A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.

A comparison of 11 beta-chloromethylestradiol and tamoxifen aziridine as affinity labeling reagents for estrogen receptors.

The tritium-labeled from of 11 beta-chloromethylestradiol was prepared by metal hydride reduction of the 17-keto derivative. Affinity labeling experiments were carried out using [3H] 11 beta-chloromethylestradiol and [3H]tamoxifen aziridine with estrogen receptor from crude, calf uterine cytosol and partially purified (heparin-sepharose chromatography) preparations. Both compounds formed highly stable receptor complexes. Estrogen specific, covalent binding, however, was indicated only for [3H]tamoxifen aziridine. An equilibrium dissociation constant of 2.8 x 10(-10) M was determined for the receptor-[3H] 11 beta-chloromethylestradiol interaction. Measurement of hormone dissociation kinetics at 30 degrees C revealed a slow, single phase dissociation of 11 beta-chloromethylestradiol from the receptor (dissociation rate constant, 1.3 x 10(-3) min-1). This contrasted with the normal biphasic dissociation pattern of estradiol in which the dissociation rate constant for the slower component was 16.7 x 10(-3) min-1. The results indicate that 11 beta-chloromethylestradiol readily converts the estrogen receptor to a high affinity binding form and suggest that the radiolabeled hormone may prove useful for studies of estrogen action.

Purification of the molybdate-stabilized 9-10 S estradiol receptor from calf uterus.

The molybdate-stabilized calf uterine estradiol receptor has been purified to near-homogeneity by a three-step procedure. Initial purification by heparin-Sepharose chromatography provides a concentrated receptor extract in 40% yield with a 5-10-fold increase in purity. The inclusion of molybdate in phosphate-buffered cytosol enhances 9-10 S receptor stability in high salt and allows elution of the oligomeric receptor complex from heparin-Sepharose with 0.4 M KCl. A second affinity step utilizing estrone carboxymethyloxime coupled to diaminoethyl bis(2-hydroxypropoxy)butane-Sepharose Cl-4B increases purification by a further 1600-fold. High performance liquid chromatography gives homogeneous receptor which migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a polypeptide of Mr approximately 89,000. The purified molybdate-stabilized receptor sediments at 9.3 +/- 0.2 S (n = 4) in glycerol gradients and has a Stokes radius of 74 +/- 3 A (n = 2) giving a calculated Mr approximately 290,000. These properties and the steroid-binding specificity of the purified receptor bear a close similarity to those found for the 9-10 S receptor in crude cytosol.

Formation of 11-trans slow reacting substances.

Under strongly basic conditions [excess LiOH, dimethoxyethane/water (4:1, vol/vol)], purified slow reacting substances (SRSs) SRS-GSH and SRS-Cys were not isomerized to their corresponding 11-trans isomers. However, addition of thiols such as glutathione (GSH) or L-cysteine to this basic medium produced various amounts of 11-trans-SRS, depending on the thiol concentration. This chemical isomerization was inhibited by the radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinooxy free radical (HTMP); the inhibition suggests that the thiyl radical (RS) is added reversibly to the triene system at C-12, resulting in the overall cis leads to trans isomerization of the 11,12 double bond. Because the amount of 11-trans-SRS-Cys produced by intact rat basophilic leukemia (RBL-1) cells was consistently higher than the amount produced in boiled cells, we believe that intact RBL-1 cells contain enzyme systems that form peroxides, which are known to enhance the formation of thiyl radicals, required for cis leads to trans isomerization. Likewise, HTMP inhibited the formation of 11-trans-SRS-Cys in this cell system.

Characterization of slow reacting substances (SRSs) of rat basophilic leukemia (RBL-1) cells: effect of cysteine on SRS profile.

When RBL-1 cells were incubated with L-cysteine (7.5 mM) and the ionophore A23187, the slow reacting substances SRS-GSH and SRS-Cys-Gly were formed. When L-cysteine was omitted in the incubation, SRS-GSH and SRS-Cys were isolated but only a trace amount of SRS-Cys-Gly was detectable. Each of the characterized SRSs was accompanied by an as yet uncharacterized structural isomer showing UV absorption at 278 nm. L-Cysteine and other thiols inhibited an aminopeptidase that transforms the highly bioactive SRS of anaphylaxis (SRS-Cys-Gly) into the less bioactive SRS-Cys. SRS-GSH, SRS-Cys-Gly, and SRS-Cys may be readily distinguished from each other by means of their bioactivities, antagonism by FPL 55712, and relative susceptibilities to the actions of soybean lipoxygenase, microsome-bound leucine aminopeptidase, and gamma-glutamyl transpeptidase.

Identification of the slow reacting substances from cat paws.

Perfusion of cat paws with compound 48/80 released two slow reacting substances (SRSs) which were isolated and characterized as 5-hydroxy-6-S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid (SRS I) and 5-hydroxy-6-S-cysteinyl-7,9,11,14-icosatetraeonic acid (SRS II) on the basis of chemical degradations, amino acid analyses, spectroscopic and enzymic experiments, and comparison with synthetic samples. The smooth muscle-contractile activities of synthetic 5-hydroxy-6-gamma-glutamylcysteinylglycyl-7,9,11,14-icosatetraenoic acid, synthetic 5-hydroxy-6-S-cysteinyl-7,9,11,14-icosatetraenoic acid, and SRS II were not inactivated by arylsulfatase. On the other hand, the spasmogenic activities produced by synthetic 5-hydroxy-6-S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid and SRS I were destroyed at the same rate by the arylsulfatase. This mode of inactivation was attributed to an aminopeptidase activity in the arylsulfatase preparation because 5-hydroxyl-6-S-cysteinyl-7,9,11,14-icosatetraenoic acid was isolated and identified as the reaction end product. Because the properties of SRS from cat paws closely resemble those of SRS generated by immunological stimulation of human tissues (SRS-A) and because all known SRS-A are inactivated by arylsulfatases, we contend that 5-hydroxy-6-S-cysteinylglycyl-7,9,11-14-icosatetraenoic acid (SRS I) corresponds to SRS-A.