Immuno-detection and differentiation of Listeria monocytogenes and Listeria ivanovii in stone fruits.

AIMS: The aim of this study was to develop a rapid detection and differentiation method for pathogenic Listeria species in stone fruits. METHODS AND RESULTS: We utilized activated charcoal enrichment media (ACM) to induce overexpression and hypersecretion of pathogenic Listeria virulence proteins which can subsequently be detected via immunoblot analysis. Plum and nectarine slices spiked with either L. monocytogenes or L. ivanovii were incubated in pre-enrichment broth followed by enrichment in ACM. Secreted proteins were precipitated and subjected to SDS-PAGE and immunoblot analysis using a combination of L. monocytogenes-specific antibody (α-listeriolysin O) and antibody specific for both L. monocytogenes and L. ivanovii (α-Internalin C). As low as 1 CFU per gram of L. monocytogenes in plum and nectarine was detected, whereas a detection limit of 10 CFU per gram was achieved for L. ivanovii in each food tested following a 20-h enrichment period. Nonpathogenic Listeria species and non-Listeria bacterial pathogens tested were negative. CONCLUSIONS: These results demonstrate the highly sensitive and specific nature of the detection method for pathogenic Listeria in stone fruits using activated charcoal enrichment as well as the capability to discriminate between L. monocytogenes and L. ivanovii. SIGNIFICANCE AND IMPACT OF THE STUDY: This method is the first to identify and differentiate L. monocytogenes and L. ivanovii in select stone fruit enrichments within 24 h using immunological techniques. The rapidity and sensitivity of the method could aid in the reduction of exposure to the public in the event of an outbreak and expedite the administration of appropriate antibiotics to infected individuals.

Bio-Plex suspension array immuno-detection of Listeria monocytogenes from cantaloupe and packaged salad using virulence protein inducing activated charcoal enrichment media.

Listeria monocytogenes, the causative agent of listeriosis in humans, is a Gram-positive bacterium that is contracted via the ingestion of contaminated foods. Two of the largest outbreaks of listeriosis occurred following consumption of tainted cantaloupe and packaged salads. Molecular methods and immuno-based techniques for detection of L. monocytogenes in these food matrices can be difficult due to the presence of assay inhibiting elements. In this study, we utilized a novel enrichment media containing activated charcoal as the key ingredient that induces hyperactive expression and secretion of L. monocytogenes virulence proteins. The Bio-Plex suspension array system, based on Luminex xMAP technology, was subsequently employed to specifically detect accumulated L. monocytogenes secreted and membrane bound proteins via paramagnetic microsphere-antibody complexes. Cantaloupe and packaged salad samples were treated with a dilution series of L. monocytogenes and incubated in activated charcoal media following a short pre-enrichment step in Buffered Listeria Enrichment Broth. Secreted L. monocytogenes lysteriolysin O was captured using magnetic microsphere-antibody conjugates and measured using the Bio-Ple×200 analyzer. As few as 100 CFU/g of L. monocytogenes was detected from both spiked cantaloupe and packaged salad samples. In addition, antibody conjugated microspheres targeting a membrane protein present on both pathogenic and nonpathogenic Listeria species was used to identify as few as 100 CFU/g of both pathogenic and nonpathogenic species in cantaloupe and packaged salad. This method presumptively identifies L. monocytogenes from cantaloupe and packaged salad in less than 24 h and non-pathogenic Listeria species within 22 h.

Real-time PCR detection of Listeria monocytogenes in infant formula and lettuce following macrophage-based isolation and enrichment.

AIMS: To develop a rapid detection procedure for Listeria monocytogenes in infant formula and lettuce using a macrophage-based enrichment protocol and real-time PCR. METHODS AND RESULTS: A macrophage cell culture system was employed for the isolation and enrichment of L. monocytogenes from infant formula and lettuce for subsequent identification using real-time PCR. Macrophage monolayers were exposed to infant formula and lettuce contaminated with a serial dilution series of L. monocytogenes. As few as approx. 10 CFU ml(-1) or g(-1) of L. monocytogenes were detected in infant formula and lettuce after 16 h postinfection by real-time PCR. Internal positive PCR controls were utilized to eliminate the possibility of false-negative results. Co-inoculation with Listeria innocua did not reduce the L. monocytogenes detection sensitivity. Intracellular L. monocytogenes could also be isolated on Listeria selective media from infected macrophage lysates for subsequent confirmation. CONCLUSIONS: The detection method is highly sensitive and specific for L. monocytogenes in infant formula and lettuce and establishes a rapid identification time of 20 and 48 h for presumptive and confirmatory identification, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is a promising alternative to many currently used q-PCR detection methods which employ traditional selective media for enrichment of contaminated food samples. Macrophage enrichment of L. monocytogenes eliminates PCR inhibitory food elements and contaminating food microflora which produce cleaner samples that increase the rapidity and sensitivity of detection.

Magnetic bead based immuno-detection of Listeria monocytogenes and Listeria ivanovii from infant formula and leafy green vegetables using the Bio-Plex suspension array system.

Listeriosis, a disease contracted via the consumption of foods contaminated with pathogenic Listeria species, can produce severe symptoms and high mortality in susceptible people and animals. The development of molecular methods and immuno-based techniques for detection of pathogenic Listeria in foods has been challenging due to the presence of assay inhibiting food components. In this study, we utilize a macrophage cell culture system for the isolation and enrichment of Listeria monocytogenes and Listeria ivanovii from infant formula and leafy green vegetables for subsequent identification using the Luminex xMAP technique. Macrophage monolayers were exposed to infant formula, lettuce and celery contaminated with L. monocytogenes or L. ivanovii. Magnetic microspheres conjugated to Listeria specific antibody were used to capture Listeria from infected macrophages and then analyzed using the Bio-Plex 200 analyzer. As few as 10 CFU/mL or g of L. monocytogenes was detected in all foods tested. The detection limit for L. ivanovii was 10 CFU/mL in infant formula and 100 CFU/g in leafy greens. Microsphere bound Listeria obtained from infected macrophage lysates could also be isolated on selective media for subsequent confirmatory identification. This method presumptively identifies L. monocytogenes and L. ivanovii from infant formula, lettuce and celery in less than 28 h with confirmatory identifications completed in less than 48 h.

Outcome of limb lengthening in fibular hemimelia and a functional foot.

BACKGROUND: The decision to recommend either reconstructive or ablative surgery to the parents of children with fibular hemimelia is difficult and debatable in the orthopaedic literature. METHODS: This is a retrospective study reporting our experience of the treatment of eight children (eight limbs) with fibular hemimelia with limb lengthening using Ilizarov or Taylor spatial frames. All of these children had type 1 or 2a fibular hemimelia (Achterman and Kalamchi). We used the number of rays present in the foot as a guide to decide on the treatment option. Children with more than three rays at the time of presentation were considered for limb reconstruction using Taylor spatial or Ilizarov frames. RESULTS: All patients were ambulatory and mobile with acceptable leg lengths and limb alignment at the time of last follow-up. All of them were satisfied with the outcome. Knee stiffness was a significant problem in the majority of the patients following lengthening. CONCLUSIONS: We conclude that limb reconstruction in children with less severe forms of fibular hemimelia is a good option.

Effect of dehydrated storage on the survival of Francisella tularensis in infant formula.

Francisella tularensis is a Gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia in humans from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, there are few studies on the long-term survivability of this organism in food matrices. Infant formula has previously been implicated as a vehicle for the transmission of a variety of bacterial pathogens in infants. In this study, we investigated the survival of F. tularensis in dehydrated infant formula under various storage conditions. F. tularensis was stored for up to 12 weeks in dehydrated infant formula in an ambient air, dry or nitrogen atmosphere. Viable counts of fresh F. tularensis at 12 weeks in infant formula revealed a 4.15, 3.37 and 3.72-log decrease in ambient air, dry and nitrogen atmosphere, respectively. D-values were calculated (in weeks) as 3.99, 4.68 and 4.47 in air, dry and nitrogen atmosphere, respectively.

Development of a macrophage cell culture method to isolate and enrich Francisella tularensis from food matrices for subsequent detection by real-time PCR.

Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia in humans from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, there are no techniques currently available to detect this organism in specific food matrices. In this study, a macrophage cell culture system is combined with real-time PCR to identify F. tularensis in food matrices. The method utilizes a mouse macrophage cell line (RAW 264.7) as host for the isolation and intracellular replication of F. tularensis. Exposure of macrophages to F. tularensis-contaminated food matrices results in uptake and intracellular replication of the bacteria, which can be subsequently detected by real-time PCR analysis of the DNA released from infected macrophage cell lysates. Macrophage monolayers were exposed to infant formula, liquid egg whites, and lettuce contaminated with varying quantities of F. tularensis. As few as 10 CFU/ml (or CFU per gram) F. tularensis was detected in infant formula and lettuce after 5 h postinfection. As few as 10 CFU/ml F. tularensis was detected in liquid egg whites after 18 h postinfection. Intracellular F. tularensis could also be isolated on Mueller-Hinton medium from lysates of macrophages infected with the bacteria in infant formula, liquid egg whites, and lettuce for subsequent confirmatory identification. This method is the first to successfully identify F. tularensis from select food matrices.

Development of a cell culture method to isolate and enrich Salmonella enterica serotype enteritidis from shell eggs for subsequent detection by real-time PCR.

Salmonella enterica serotype Enteritidis is a major cause of nontyphoidal salmonellosis from ingestion of contaminated raw or undercooked shell eggs. Current techniques used to identify Salmonella serotype Enteritidis in eggs are extremely laborious and time-consuming. In this study, a novel eukaryotic cell culture system was combined with real-time PCR analysis to rapidly identify Salmonella serotype Enteritidis in raw shell eggs. The system was compared to the standard microbiological method of the International Organization for Standardization (Anonymous, Microbiology of food and animal feeding stuffs-horizontal method for the detection of Salmonella, 2002). The novel technique utilizes a mouse macrophage cell line (RAW 264.7) as the host for the isolation and intracellular replication of Salmonella serotype Enteritidis. Exposure of macrophages to Salmonella serotype Enteritidis-contaminated eggs results in uptake and intracellular replication of the bacterium, which can subsequently be detected by real-time PCR analysis of the DNA released after disruption of infected macrophages. Macrophage monolayers were exposed to eggs contaminated with various quantities of Salmonella serotype Enteritidis. As few as 10 CFU/ml was detected in cell lysates from infected macrophages after 10 h by real-time PCR using primer and probe sets specific for DNA segments located on the Salmonella serotype Enteritidis genes sefA and orgC. Salmonella serotype Enteritidis could also be distinguished from other non-serogroup D Salmonella serotypes by using the sefA- and orgC-specific primer and probe sets. Confirmatory identification of Salmonella serotype Enteritidis in eggs was also achieved by isolation of intracellular bacteria from lysates of infected macrophages on xylose lysine deoxycholate medium. This method identifies Salmonella serotype Enteritidis from eggs in less than 10 h compared to the more than 5 days required for the standard reference microbiological method of the International Organization for Standardization (Microbiology of food and animal feeding stuffs-horizontal method for the detection of Salmonella, 2002).

Thermal resistance of Francisella tularensis in infant formula and fruit juices.

Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, little information exists on the thermal stability of this organism in food matrices. In the present study, the thermal resistance of the live vaccine strain of F. tularensis in four food products (liquid infant formula, apple juice, mango juice, and orange juice) was investigated. D-values ranged from 12 s (57.5 degrees C) to 580 s (50 degrees C) in infant formula with a z-value of 4.37 degrees C. D-values in apple juice ranged from 8 s (57.5 degrees C) to 59 s (50 degrees C) with a z-value of 9.17 degrees C. The live vaccine strain did not survive at temperatures above 55 degrees C in mango juice and orange juice (>6-log inactivation). D-values at 55 to 47.5 degrees C were 15 to 59 s in mango juice and 16 to 105 s in orange juice with z-values of 9.28 and 12.30 degrees C, respectively. These results indicate that current pasteurization parameters used for destroying common foodborne bacterial pathogens are adequate for eliminating F. tularensis in the four foods tested. This study is the first to determine thermal inactivation of F. tularensis in specific foods and will permit comparisons with the thermal inactivation data of other more traditional foodborne pathogens.

The ferguson medial approach for open reduction of developmental dysplasia of the hip. A clinical and radiological review of 49 hips.

The results of the Ferguson medial approach for open reduction of developmental dysplasia of the hip (DDH) were reviewed for 49 hips with a follow-up of more than 48 months. The mean age at operation was 12.3 months (6 to 23). The mean length of clinical and radiological follow-up was 82 months (48 to 148). Three redislocations occurred. Group I avascular necrosis according to the classification of Kalamchi and MacEwen was seen in four hips, group II in two hips and group III in one hip; 92% of the hips were classified as Severin class I and II. The acetabular index and centre edge (CE) angles were within normal limits at final follow-up, but were still significantly different from the unaffected side. We conclude that the Ferguson procedure is safe and reliable for low dislocations in children aged six to 18 months.

Type III export: new uses for an old pathway.

Gram-negative bacteria use type III secretion (TTS) systems to translocate proteins into the extracellular environment or directly into eukaryotic cells. These complex secretory systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway. Secretion substrates are directed to the TTS machinery via mRNA and/or amino acid secretion signals. TTS chaperones bind to select secretion substrates and assist in the export process. Recent progress in the understanding of TTS is reviewed.

Yersinia pestis YscG protein is a Syc-like chaperone that directly binds yscE.

Pathogenic Yersinia species secrete virulence proteins, termed Yersinia outer proteins (Yops), upon contact with a eukaryotic cell. The secretion machinery is composed of 21 Yersinia secretion (Ysc) proteins. Yersinia pestis mutants defective in expression of YscG or YscE were unable to export the Yops. YscG showed structural and limited amino-acid-sequence similarities to members of the specific Yop chaperone (Syc) family of proteins. YscG specifically recognized and bound YscE; however, unlike previously characterized Syc substrates, YscE was not exported from the cell. These data suggest that YscG functions as a chaperone for YscE.

The Yersinia pestis YscY protein directly binds YscX, a secreted component of the type III secretion machinery.

Human pathogenic yersiniae organisms export and translocate the Yop virulence proteins and V antigen upon contact with a eukaryotic cell. Yersinia pestis mutants defective for production of YscX or YscY were unable to export the Yops and V antigen. YscX and YscY were both present in the Y. pestis cell pellet fraction; however, YscX was also found in the culture supernatant. YscY showed structural and amino acid sequence similarities to the Syc family of proteins. YscY specifically recognized and bound to a region of YscX that included a predicted coiled-coil region. These data suggest that YscY may function as a chaperone for YscX in Y. pestis.

YscB of Yersinia pestis functions as a specific chaperone for YopN.

Following contact with a eucaryotic cell, Yersinia species pathogenic for humans (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) export and translocate a distinct set of virulence proteins (YopE, YopH, YopJ, YopM, and YpkA) from the bacterium into the eucaryotic cell. During in vitro growth at 37 degrees C in the presence of calcium, Yop secretion is blocked; however, in the absence of calcium, Yop secretion is triggered. Yop secretion occurs via a plasmid-encoded type III, or "contact-dependent," secretion system. The secreted YopN (also known as LcrE), TyeA, and LcrG proteins are necessary to prevent Yop secretion in the presence of calcium and prior to contact with a eucaryotic cell. In this paper we characterize the role of the yscB gene product in the regulation of Yop secretion in Y. pestis. A yscB deletion mutant secreted YopM and V antigen both in the presence and in the absence of calcium; however, the export of YopN was specifically reduced in this strain. Complementation with a functional copy of yscB in trans completely restored the wild-type secretion phenotype for YopM, YopN, and V antigen. The YscB amino acid sequence showed significant similarities to those of SycE and SycH, the specific Yop chaperones for YopE and YopH, respectively. Protein cross-linking and immunoprecipitation studies demonstrated a specific interaction between YscB and YopN. In-frame deletions in yopN eliminating the coding region for amino acids 51 to 85 or 6 to 100 prevented the interaction of YopN with YscB. Taken together, these results indicate that YscB functions as a specific chaperone for YopN in Y. pestis.

A complex composed of SycN and YscB functions as a specific chaperone for YopN in Yersinia pestis.

Human pathogenic Yersinia resist host defences, in part through the expression and delivery of a set of plasmid-encoded virulence proteins termed Yops. A number of these Yops are exported from the bacteria directly into the cytoplasm of their eukaryotic host's cells upon contact with these cells. The secreted YopN protein (also known as LcrE) is required to block Yop secretion in the presence of calcium in vitro or before contact with a eukaryotic cell in vivo. In this study, we characterize the role of the tyeA, sycN and yscB gene products in the regulation of Yop secretion in Yersinia pestis. Mutants specifically defective in the expression of TyeA, SycN or YscB were no longer able to block Yop secretion in the presence of calcium. In addition, the secretion of YopN was specifically reduced in both the sycN and the yscB deletion mutants. Protein cross-linking and immunoprecipitation studies in conjunction with yeast two-hybrid analyses showed that SycN and YscB interact with one another to form a SycN/YscB complex. Yeast three-hybrid analyses demonstrated that the SycN/YscB complex, but not SycN or YscB alone, specifically associates with YopN. SycN and YscB share amino acid sequence similarity and structural similarities with the specific Yop chaperones SycE and SycH. Together, these results indicate that a complex composed of SycN and YscB functions as a specific chaperone for YopN in Y. pestis.

A comparative study of sheep and pigs given the tremorgenic mycotoxins verruculogen and penitrem A.

The moulds Penicillium simplicissimum and P crustosum and the tremorgenic mycotoxins, verruculogen and penitrem A, isolated from them, were given to sheep and pigs to compare their potencies. Pigs were generally less susceptible and in both species penitrem A was less potent than verruculogen. Five-month-old lambs seemed more susceptible to mycelium containing verruculogen than were 15-month-old sheep given a similar oral dose relative to bodyweight. Repeated daily dosing of lambs and sheep for five days with P simplicissimum failed to enhance the effect, indicating that verruculogen toxicity was not cumulative. Long and short acting barbiturate anaesthesia blocked the effects of lethal doses of tremorgens. Sedation with diazepam diminished, but did not block, mycotoxin-induced tremors suggesting that there was no specific action of this anticonvulsant sedative on tremorgens.

Metabolism of verruculogen in rats.

Radiolabeled verruculogen was detected in a wide range of body tissues 6 min after intravenous administration, but after a further 20 min it was mainly being excreted via the biliary route. In isolated liver perfusion, [14C]verruculogen was rapidly taken up by the liver and metabolized completely, principally to the related tremorgen TR-2 but also to a desoxy derivative of verruculogen. In addition, a smaller amount of an isomer of TR-2 was detected. These metabolic products were excreted in the bile.