Roberta J. Magnuson

Use of Metagenomic Shotgun Sequencing Technology To Detect Foodborne Pathogens within the Microbiome of the Beef Production Chain

ABSTRACT Foodborne illnesses associated with pathogenic bacteria are a global public health and economic challenge. The diversity of microorganisms (pathogenic and nonpathogenic) that exists within the food and meat industries complicates efforts to understand pathogen ecology. Further, little is known about the interaction of pathogens within the microbiome throughout the meat production chain. Here, a metagenomic approach and shotgun sequencing technology were used as tools to detect pathogenic bacteria in environmental samples collected from the same groups of cattle at different longitudinal processing steps of the beef production chain: cattle entry to feedlot, exit from feedlot, cattle transport trucks, abattoir holding pens, and the end of the fabrication system. The log read counts classified as pathogens per million reads for Salmonella enterica, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Clostridium spp. (C. botulinum and C. perfringens), and Campylobacter spp. (C. jejuni, C. coli, and C. fetus) decreased over subsequential processing steps. Furthermore, the normalized read counts for S. enterica, E. coli, and C. botulinum were greater in the final product than at the feedlots, indicating that the proportion of these bacteria increased (the effect on absolute numbers was unknown) within the remaining microbiome. From an ecological perspective, data indicated that shotgun metagenomics can be used to evaluate not only the microbiome but also shifts in pathogen populations during beef production. Nonetheless, there were several challenges in this analysis approach, one of the main ones being the identification of the specific pathogen from which the sequence reads originated, which makes this approach impractical for use in pathogen identification for regulatory and confirmation purposes.

Resistome diversity in cattle and the environment decreases during beef production

Antimicrobial resistant determinants (ARDs) can be transmitted from livestock systems through meat products or environmental effluents. The public health risk posed by these two routes is not well understood, particularly in non-pathogenic bacteria. We collected pooled samples from 8 groups of 1741 commercial cattle as they moved through the process of beef production from feedlot entry through slaughter. We recorded antimicrobial drug exposures and interrogated the resistome at points in production when management procedures could potentially influence ARD abundance and/or transmission. Over 300 unique ARDs were identified. Resistome diversity decreased while cattle were in the feedlot, indicating selective pressure. ARDs were not identified in beef products, suggesting that slaughter interventions may reduce the risk of transmission of ARDs to beef consumers. This report highlights the utility and limitations of metagenomics for assessing public health risks regarding antimicrobial resistance, and demonstrates that environmental pathways may represent a greater risk than the food supply. DOI: http://dx.doi.org/10.7554/eLife.13195.001

Characterization of the resistome in manure, soil and wastewater from dairy and beef production systems.

It has been proposed that livestock production effluents such as wastewater, airborne dust and manure increase the density of antimicrobial resistant bacteria and genes in the environment. The public health risk posed by this proposed outcome has been difficult to quantify using traditional microbiological approaches. We utilized shotgun metagenomics to provide a first description of the resistome of North American dairy and beef production effluents, and identify factors that significantly impact this resistome. We identified 34 mechanisms of antimicrobial drug resistance within 34 soil, manure and wastewater samples from feedlot, ranch and dairy operations. The majority of resistance-associated sequences found in all samples belonged to tetracycline resistance mechanisms. We found that the ranch samples contained significantly fewer resistance mechanisms than dairy and feedlot samples, and that the resistome of dairy operations differed significantly from that of feedlots. The resistome in soil, manure and wastewater differed, suggesting that management of these effluents should be tailored appropriately. By providing a baseline of the cattle production waste resistome, this study represents a solid foundation for future efforts to characterize and quantify the public health risk posed by livestock effluents.

Lack of correlation between antibody titers to fibrinogen-binding protein of Streptococcus equi and persistent carriers of strangles

Previously published studies have neither used nor reported the results of an indirect enzyme-linked immunosorbent assay (iELISA) to measure serologic responses in natural outbreaks of strangles. The concept of using serologic responses to identify persistent carriers of Streptococcus equi has been proposed but not scientifically evaluated. The specific aims of the current study were to determine the duration and level of truncated fibrinogen-binding protein-specific (SeM allele 1) antibody production in ponies involved in a natural outbreak of strangles and to determine if test results from this serologic iELISA could predict persistent carrier status. Serologic samples were obtained before and after an outbreak of naturally occurring strangles infection. Persistent carriers of S. equi were identified via culture and polymerase chain reaction (PCR) testing of lavage fluid collected from the guttural pouches and nasopharynx or swabs of the nasopharynx after recovery from acute disease and at postmortem examination. Logistic regression analysis was used to determine if an association existed between serologic response and persistent carrier state. The ELISA reported in the current study definitively confirmed a recent exposure to S. equi. However, the measured serologic response did not predict carrier status in this strangles outbreak. Therefore, a guttural-pouch endoscopy with subsequent culture or PCR testing to detect S. equi remains the most accurate method available for the identification of persistent carriers.

Adsorptive effects of di-tri-octahedral smectite on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies

OBJECTIVE To determine the adsorptive capability of di-tri-octahedral smectite (DTOS) on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies. SAMPLE POPULATION 3 C perfringens exotoxins and 9 colostral samples. PROCEDURES Alpha, beta, and beta-2 exotoxins were individually co-incubated with serial dilutions of DTOS or bismuth subsalicylate, and the amount of toxin remaining after incubation was determined via toxin-specific ELISAs. Colostral samples from healthy mares were individually co-incubated with serial dilutions of DTOS, and colostral IgG concentrations were determined via single radial immunodiffusion assay. RESULTS Di-tri-octahedral smectite decreased the amount of each C perfringens exotoxin in co-incubated samples in a dose-dependent manner and was more effective than bismuth subsalicylate at reducing exotoxins in vitro. Decreases in the concentration of IgG were detected in samples of colostrum that were combined with DTOS at 1:4 through 1:16 dilutions, whereas no significant decrease was evident with DTOS at the 1:32 dilution. CONCLUSIONS AND CLINICAL RELEVANCE Di-tri-octahedral smectite effectively adsorbed C perfringens exotoxins in vitro and had a dose-dependent effect on the availability of equine colostral antibodies. Results suggested that DTOS may be an appropriate adjunctive treatment in the management of neonatal clostridiosis in horses. In vivo studies are necessary to fully assess the clinical efficacy of DTOS treatment.

A single-tube multiplex reverse transcription-polymerase chain reaction for detection and differentiation of vesicular stomatitis Indiana 1 and New Jersey viruses in insects

A multiplex single-tube reverse transcription—polymerase chain reaction (RT-PCR) has been developed for the detection and differentiation of vesicular stomatitis viruses (VSV), Indiana 1 and New Jersey, from insect samples. Using this assay, detection of either or both viruses in as little as 20 fg of total RNA from tissue culture was achieved, along with detection of vesicular stomatitis (VS) RNA from macerates containing 2 infected mosquitoes in pools of 10—30 noninfected mosquitoes. Vesicular stomatitis virus was detected by RT-PCR in all culture-positive samples, and detection as low as 4 plaque forming units per milliliter was achieved. Comparison between RT-PCR and tissue culture revealed that RT-PCR was able to detect VSV in a volume of insect macerate averaging almost 100 times less than that required for detection by tissue culture. The reported RT-PCR is a potential valuable tool for rapid and sensitive detection and differentiation of VS in insects because intense work associated with viral isolation, the cytotoxicity of insect extracts, and separate virus identification steps can be avoided. Potential application to detection and differentiation of VSV serotypes from vertebrate hosts is addressed.

Prevalence of Escherichia coli O157 Shedding in Preweaned Calves on Colorado Dairies

To gain insight into a potential age-related predisposition for Escherichia coli pathogen shedding on dairies, this pilot study measured the prevalence of E. coli O157 (ECO157) in the feces of preweaned dairy calves. An aim of this study was to link these outcomes with the concurrent environmental presence of ECO157 and dam ECO157 shedding elucidated in a parallel study. Recto-anal mucosal swabs and a subset of fecal grab samples were collected from calves (2 to 8 weeks of age; n = 399) monthly between December 2013 and June 2014 on three dairies in northern Colorado. A subset of calf dams (n = 111) were also sampled via fecal grab. Concurrently, environmental samples were collected from locations within the vicinity of the calves: farm tractor tires, steering wheels, hutches, buckets, and gloves from the research technicians and the employees involved in calf rearing. The presence of ECO157 and virulence genes was measured in the samples and confirmed via PCR. Of the calves, only 1 (0.25%) of 399 individuals shed during the time period, and the ECO157 strain detected carried no measured virulence genes (eaeA, stx1, and stx2). No difference was seen in detection between the recto-anal mucosal swabs and the fecal grab technique. In contrast, 32% (35 of 111) of the dams shed ECO157, with 1.8% (2 of 111) of the shed isolates containing virulence genes. No ECO157 was detected in the environmental samples. These outcomes demonstrate a disparity between dam and calf ECO157 shedding and indicate that preweaned calves, managed similarly to those of this study, probably have a minor influence on dairy contamination and the transmission of ECO157.

Antimicrobial Resistance Profiles in Escherichia coli O157 Isolates from Northern Colorado Dairies

Escherichia coli O157 (EcO157) infections can lead to serious disease and death in humans. Although the ecology of EcO157 is complex, ruminant animals serve as an important reservoir for human infection. Dairy cattle are unique because they may be a source of contamination for milk, meat, and manure-fertilized crops. Foodborne dairy pathogens such as EcO157 are of primary importance to public health. Antimicrobial resistance (AMR) is a complex phenomenon that complicates the treatment of serious bacterial infections and is of increasing concern. In the face of recommended use restrictions for antimicrobial agents in livestock operations, current AMR patterns in known foodborne pathogens should be documented. The objective of this study was to document AMR patterns in EcO157 isolates from dairies in northern Colorado using antimicrobial agents commonly found on dairies and representative of medically important antimicrobial drug classes. Seventy-five EcO157 isolates were recovered from three dairies. Six isolates were resistant to at least 1 of the 10 tested antimicrobial agents: four were resistant to streptomycin, sulfisoxazole, and tetracycline; one was resistant to streptomycin and tetracycline; and one was resistant to only tetracycline. All resistant isolates were from a single dairy. Overall, a low prevalence (8%) of AMR was observed among the 75 EcO157 isolates. No significant effects on AMR profiles due to virulence genes, parity, or previous antimicrobial treatments within the current lactation period were detected. The results of this study provide background information for future comparative studies investigating AMR trends. Future studies should include more participating farms and more samples and should control for potential confounding factors of AMR that may underlie individual farm variation.

Inhibiting avian influenza virus shedding using a novel RNAi antiviral vector technology: proof of concept in an avian cell model

Influenza A viruses pose significant health and economic threats to humans and animals. Outbreaks of avian influenza virus (AIV) are a liability to the poultry industry and increase the risk for transmission to humans. There are limitations to using the AIV vaccine in poultry, creating barriers to controlling outbreaks and a need for alternative effective control measures. Application of RNA interference (RNAi) techniques hold potential; however, the delivery of RNAi-mediating agents is a well-known obstacle to harnessing its clinical application. We introduce a novel antiviral approach using bacterial vectors that target avian mucosal epithelial cells and deliver (small interfering RNA) siRNAs against two AIV genes, nucleoprotein (NP) and polymerase acidic protein (PA). Using a red fluorescent reporter, we first demonstrated vector delivery and intracellular expression in avian epithelial cells. Subsequently, we demonstrated significant reductions in AIV shedding when applying these anti-AIV vectors prophylactically. These antiviral vectors provided up to a 10,000-fold reduction in viral titers shed, demonstrating in vitro proof-of-concept for using these novel anti-AIV vectors to inhibit AIV shedding. Our results indicate this siRNA vector technology could represent a scalable and clinically applicable antiviral technology for avian and human influenza and a prototype for RNAi-based vectors against other viruses.

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one‐step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine‐thiocyanate‐based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column‐based purification coupled with one‐step RT‐PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri‐phasic extraction method (Tri‐reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT‐PCR was as low as 0.505 to 2.84 TCID50 for VSV‐IND and VSV‐NJ, respectively. The multiplex RT‐PCR consistently detected VSV‐IND and NJ RNA in as little as 0.1–1.0 fg of total RNA from spiked BHK‐21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT‐PCR assay was capable of detecting both types of VSV in a one‐step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID50 (IND), 0.0946 TCID50 (NJ), and 0.057 fg (IND and NJ) per 2 µl PCR sample, which is significantly more sensitive than reported previously (0.28–2.8 TCID50/1 µl). So the present study improved the sensitivity of previously reported multiplex RT‐PCR for the detection and differentiation of VSV‐IND and VSV‐NJ in a single assay. J. Clin. Lab. Anal. 25:95–99, 2011.