PK-sensitive PrP is infectious and shares basic structural features with PK-resistant PrP.

One of the main characteristics of the transmissible isoform of the prion protein (PrP(Sc)) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP(Sc) following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP(Sc) that is sensitive to PK hydrolysis (sPrP(Sc)). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP(Sc) versus the PK-resistant (rPrP(Sc)) fractions of PrP(Sc) and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP(Sc) and rPrP(Sc) fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP(Sc) to the rPrP(Sc) forms of PrP(Sc). Although the sPrP(Sc) and rPrP(Sc) fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.

Effects of Antimicrobial Interventions on Indicator Organisms during Beef Carcass Dressing.

ABSTRACT: Beef slaughter establishments employ many interventions to help minimize the occurrence of pathogens in their products. This study explored the effectiveness of various common interventions on microbial load using the results of the Beef-Veal Carcass Baseline Survey conducted in 2014 to 2015. The Food Safety and Inspection Service analyzed swab samples taken from 1,135 carcasses at 139 establishments. These included paired samples from post-hide removal (before evisceration) and prechill (after evisceration). Samples were tested for pathogens (Salmonella and Shiga toxin-producing Escherichia coli) and indicators (E. coli, Enterobacteriaceae, coliforms, and aerobic count [AC]). The sample size for pathogen-positive samples was small, impeding the establishment of a direct correlation between interventions and pathogens. However, we observed associations between pathogen-positive rate and log AC, indicating similar intervention effectiveness of pathogens and indicators in this study. Generally, the use of interventions reduced indicator concentrations. Each intervention produced a range of effectiveness, suggesting that how interventions are applied may be as important as which interventions are applied. The range of effectiveness for single interventions was a 0.4- to 1.9-log AC reduction; for multihurdle interventions, it ranged from 1.6- to 2.9-log AC reduction. The results of this study may be used by slaughter establishments to help identify effective intervention options for pathogen reduction.

Effects of purification on the bioavailability of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. They are primarily produced by the gram-positive, anaerobic spore-forming bacterium, Clostridium botulinum. In bacterial cultures, secreted BoNTs are associated with non-toxic accessory proteins forming large complexes. Neurotoxin-associated proteins have been shown to play an important role in the oral toxicity of BoNTs by protecting them from degradation and digestion by gastric acid and enzymes. Most toxicity studies using BoNTs have been performed using highly purified toxin. In this study, the toxicities of purified and crude BoNT/A toxin preparations were compared. Protein components secreted into culture supernatants along with BoNT/A were identified by mass spectrometry and the contribution of extra proteins found in the soluble crude toxin extracts to the toxicity of BoNTs was determined in mouse models of oral and parenteral botulinum intoxication. Analysis of crude toxin composition permitted assessment of the impact of accessory proteins on the oral bioavailability of BoNT/A toxin in food matrices.

Multiplexed Microsphere Suspension Array-Based Immunoassays.

ELISA is an extremely powerful tool to detect analytes because of its sensitivity, selectivity, reproducibility and ease of use. Here we describe sandwich immunoassays performed in suspension on spectrally unique microspheres developed by Luminex. Luminex assays offer the benefit of multiplex analysis of large numbers of analytes in a single reaction. Because the microspheres are spectrally unique, many microspheres, each attached to various antibodies, can be added to a single sample. Luminex instruments can distinguish each microsphere and detect the intensity of a reporter signal for each microsphere. Results are reported in Median Fluorescent Intensities for each analyte. Luminex assays can be used to detect up to 500 analytes in a high-throughput format. Luminex refers to this technology as xMAP(®). Here we describe a routine protocol for a Luminex immunoassay. Other Luminex assays would have to be optimized for specific conditions according to their use.

Prediction of B-Cell Epitopes in Listeriolysin O, a Cholesterol Dependent Cytolysin Secreted by Listeria monocytogenes.

Listeria monocytogenes is a gram-positive, foodborne bacterium responsible for disease in humans and animals. Listeriolysin O (LLO) is a required virulence factor for the pathogenic effects of L. monocytogenes. Bioinformatics revealed conserved putative epitopes of LLO that could be used to develop monoclonal antibodies against LLO. Continuous and discontinuous epitopes were located by using four different B-cell prediction algorithms. Three-dimensional molecular models were generated to more precisely characterize the predicted antigenicity of LLO. Domain 4 was predicted to contain five of eleven continuous epitopes. A large portion of domain 4 was also predicted to comprise discontinuous immunogenic epitopes. Domain 4 of LLO may serve as an immunogen for eliciting monoclonal antibodies that can be used to study the pathogenesis of L. monocytogenes as well as develop an inexpensive assay.

A 7-plex microbead-based immunoassay for serotyping Shiga toxin-producing Escherichia coli.

Serotyping of Shiga toxin-producing Escherichia coli (STEC) has been contingent upon the availability of antisera. Here we describe a 7-plex microbead-based immunoassay to simultaneously serotype seven STECs (i.e., belonging to serogroups O26, O45, O103, O111, O121, O145, and O157) by the Luminex xMAP® technology. This technology presents many advantages: Its multiplexed format (up to 100 analytes) saves time, reagents, and test sample, and many regulatory agencies currently utilize this platform for other assays. In this study, a total of seventy-nine STEC strains belonging to the 7 different serogroups of interest were tested. These strains had been previously serotyped and their serogroup was confirmed by PCR. Except for one strain belonging to the O111 serogroup, nearly all strains (i.e., 98.7%; 78/79) were correctly identified on the Bio-Plex 100 instrument in less than 4h. This newly developed microbead-based immunoassay could be extended to include other STEC serogroups, virulence factors, and/or bacterial species.

Dynex: multiplex ELISA technology.

Conventional enzyme-linked immunosorbent assay (ELISA) is a gold standard for screening antibodies and testing for protein or antigen presence. A significant limitation of this assay resides in the fact that only one analyte can be assessed per microplate well. Here, we describe and investigate a new technology consisting of an automated ELISA system in which up to 10 analytes can be measured within one single well, thus improving productivity, accuracy, and repeatability by reducing the amount of human labor required. Another strength of the platform is that a user can load any necessary sets/subsets of beads to perform required assays, with improved flexibility compared to manufactured-loaded arrays for multiplex analysis. We also demonstrate that this system can be used to determine the pathogenicity (i.e., presence of Shiga toxins) and serotype (i.e., Escherichia coli O157) of E. coli isolates.

Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods.

Shiga toxin-producing Escherichia coli (STEC) is a significant foodborne pathogen with great economic consequences. There has been an increased food safety concern with this organism since outbreaks of human illnesses caused by this pathogen were first reported in 1982. Therefore, developing a reliable, sensitive, and rapid assay capable of detecting E. coli O157 and the main toxins produced by STEC (i.e., Shiga toxins 1 [Stx(1)] and 2 [Stx(2)]) will directly benefit regulatory agencies by minimizing analysis time. Here, we use Luminex technology to detect multiple analytes in a single 50-ml sample. Using commercially available monoclonal antibodies coupled to carboxylated magnetic microbeads, we developed an immunoassay capable of simultaneously serotyping E. coli O157 and detecting Stx(1) and/or Stx(2). The specificity and sensitivity of this immunoassay was tested against a collection of 34 E. coli isolates belonging to various O serogroups phenotypically different for Stx. The results were compared with microplate sandwich enzyme-linked immunosorbent assay (ELISA), and no cross-reactivity was observed for any of the monoclonal antibodies used. An increased sensitivity up to 1,000 times was observed in the microbead-based immunoassay when compared with the microplate sandwich ELISA. The results indicate that Luminex technology has the potential to simultaneously detect multiple targets without loss of specificity and/or sensitivity. A blind experiment was conducted with 48 samples of ground beef, lettuce, and milk spiked with ≤2 CFU/g E. coli. All the samples were correctly identified, with no false positives or false negatives. This microbead-based immunoassay could be extended to simultaneously detect additional foodborne pathogens and their toxic markers.

Rapid O serogroup identification of the ten most clinically relevant STECs by Luminex microbead-based suspension array.

Identification and serotyping of Shiga toxin-producing Escherichia coli during foodborne outbreaks can aid in matching clinical, food, and environmental isolates when trying to identify the source of illness and ultimately food contamination. Herein we describe a Luminex microbead-based suspension array to identify the O serogroup of the ten most clinically relevant STECs: O26, O45, O91, O103, O111, O113, O121, O128, O145, and O157. The use of PCR followed by Luminex xMAP® technology enables the detection of multiple analytes in a single multiplex reaction with high throughput capabilities. One hundred and fourteen STEC isolates were correctly identified with no false positives among forty-six other organisms using this assay. Assay performance was tested in multiple laboratories using a panel of eleven different STEC serogroups on the Bio-Plex 200 and MAGPIX instruments. The STEC microbead-based suspension array can be performed in a 96-well plate format for high throughput screening in less than 4h. Furthermore, it is expandable, allowing for the addition of O serogroups should the need arise.