Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

Detection and Isolation of Campylobacter spp. from Raw Meat.

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 µm and 0.65 µm pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 µm pore-size filter compared to the 0.45 µm pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.

Use of a commercial tissue dissociation system to detect Salmonella-contaminated poultry products.

Successful detection of bacterial pathogens in food can be challenging due to the physical and compositional complexity of the matrix. Different mechanical/physical and chemical methods have been developed to separate microorganisms from food matrices to facilitate detection. The present study benchmarked a commercial tissue digestion system that applies both chemical and physical methods to separate microorganisms from tissues against stomaching, a standard process currently utilized by commercial and regulatory food safety laboratories. The impacts of the treatments on the physical properties of the food matrix were characterized along with the compatibility of the methods with downstream microbiological and molecular detection assays. The results indicate the tissue digestion system can significantly reduce the average particle size of the chicken sample relative to processing via a stomacher (P < 0.001) without adversely affecting either real-time PCR (qPCR) or plate counting assays, which are typically used to detect Salmonella. Furthermore, inoculated chicken treated with the GentleMACS resulted in a significant increase (P < 0.003) in the qPCR's detection capabilities relative to stomached controls. Cohen kappa (κ) coefficient and McNemar's test indicate the plating assays and PCR results agree with measurements obtained via the 3 M Molecular Detection System as defined in the MLG standard (κ > 0.62; P > 0.08). Collectively, the results demonstrate that the technique enables detection of pathogens in meat at lower levels of contamination using current industry standard technologies.

Photo-Enhanced Oil Toxicity to Alcid Immune Function.

Oil spills are devastating to seabirds, causing high levels of mortality and toxic physiological effects, especially to immune function. Sunlight exposure can further enhance the toxicity of oil to marine species by generating photodegradation products. Photo-enhanced oil toxicity to marine birds has not been studied. Therefore, the goal of the present study was to investigate the toxicity and photo-enhanced toxicity of oil to lymphocyte proliferation, macrophage phagocytosis, and reactive oxygen species production in three alcid species, common murres (Uria aalge), tufted puffins (Fratercula cirrhata), and horned puffins (Fratercula corniculata). Intrinsic factors (species, age, and sex) had a more significant effect on lymphocyte proliferation than exposure to oil or photoactivated oil. Macrophage phagocytosis was significantly reduced in oil and photoactivated oil treatments, whereas hydrogen peroxide production was significantly increased. Interestingly, nonphotoactivated oil stimulated significantly more hydrogen peroxide than photoactivated oil. The results suggest that alcid immune function could be variably influenced during an oil spill depending on the species, sex, and age of the bird as well as the season and level of sunlight exposure. Environ Toxicol Chem 2023;42:2701-2711. © 2023 SETAC.

Evaluation of Long-Read Sequencing Simulators to Assess Real-World Applications for Food Safety.

Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes are routinely responsible for severe foodborne illnesses in the United States. Current identification methods utilized by the U.S. Food Safety Inspection Service require at least four days to identify STEC and six days for L. monocytogenes. Adoption of long-read, whole genome sequencing for food safety testing could significantly reduce the time needed for identification, but method development costs are high. Therefore, the goal of this project was to use NanoSim-H software to simulate Oxford Nanopore sequencing reads to assess the feasibility of sequencing-based foodborne pathogen detection and guide experimental design. Sequencing reads were simulated for STEC, L. monocytogenes, and a 1:1 combination of STEC and Bos taurus genomes using NanoSim-H. At least 2500 simulated reads were needed to identify the seven genes of interest targeted in STEC, and at least 500 reads were needed to detect the gene targeted in L. monocytogenes. Genome coverage of 30x was estimated at 21,521, and 11,802 reads for STEC and L. monocytogenes, respectively. Approximately 5-6% of reads simulated from both bacteria did not align with their respective reference genomes due to the introduction of errors. For the STEC and B. taurus 1:1 genome mixture, all genes of interest were detected with 1,000,000 reads, but less than 1x coverage was obtained. The results suggested sample enrichment would be necessary to detect foodborne pathogens with long-read sequencing, but this would still decrease the time needed from current methods. Additionally, simulation data will be useful for reducing the time and expense associated with laboratory experimentation.

Gene Expression Profiles in Two Razor Clam Populations: Discerning Drivers of Population Status.

With rapidly changing marine ecosystems, shifts in abundance and distribution are being documented for a variety of intertidal species. We examined two adjacent populations of Pacific razor clams (Siliqua patula) in lower Cook Inlet, Alaska. One population (east) supported a sport and personal use fishery, but this has been closed since 2015 due to declines in abundance, and the second population (west) continues to support commercial and sport fisheries. We used gene expression to investigate potential causes of the east side decline, comparing razor clam physiological responses between east and west Cook Inlet. The target gene profile used was developed for razor clam populations in Alaska based on physiological responses to environmental stressors. In this study, we identified no differences of gene expression between east and west populations, leading to two potential conclusions: (1) differences in factors capable of influencing physiology exist between the east and west and are sufficient to influence razor clam populations but are not detected by the genes in our panel, or (2) physiological processes do not account for the differences in abundance, and other factors such as predation or changes in habitat may be impacting the east Cook Inlet population.

Competency of common northern sea otter (Enhydra lutris kenyoni) prey items to harbor Streptococcus lutetiensis and S. phocae.

Streptococcus lutetiensis and S. phocae have been associated with significant morbidity and mortality in northern sea otters Enhydra lutris kenyoni in Alaska, USA, but the route and mechanism(s) of transmission remain unknown. The goal of this study was to determine the competence of common northern sea otter prey to harbor 2 species of pathogenic Streptococcus bacteria. Prey items (bay mussels Mytilus trossulus, butter clams Saxidomus giganteus, Dungeness crab Metacarcinus magister and black turban snails Tegula funebralis) were exposed to known concentrations of exponential phase cultures of S. lutetiensis and S. phocae in seawater for 24 h. A quantitative PCR assay was developed targeting the sodA gene of both S. lutetiensis and S. phocae to quantify DNA in the prey samples. Results (mean ± SD) revealed that butter clams had the highest concentration of bacteria (4.32 × 107 ± 8.20 × 106 CFU ml-1 of S. lutetiensis, 1.20 × 108 ± 2.08 × 107 CFU ml-1 of S. phocae), followed by mussels (4.26 × 107 ± 1.66 × 107 CFU ml-1, 1.16 × 108 ± 5.39 × 107 CFU ml-1), snails (1.90 × 107 ± 5.26 × 106 CFU ml-1, 5.97 × 107 ± 2.07 × 107 CFU ml-1) and crab (1.46 × 107 ± 0 CFU ml-1, 1.64 × 107 ± 0 CFU ml-1). All prey species harbored higher concentrations of S. phocae than S. lutetiensis.

Differential Progression of Lymphoma in Two Captive Steller's Eiders (Polysticta stelleri).

This report details 2 different presentations of lymphoma in captive Steller's eiders (Polysticta stelleri). A female Steller's eider, at least 10 years old, developed lameness and lethargy. A complete blood cell count (CBC) revealed a severely elevated total white blood cell (WBC) count with a lymphocytosis. A subsequent liver biopsy confirmed a diagnosis of lymphoma and lymphocytic leukemia. The female Steller's eider was euthanatized 11 days after presentation. On necropsy, neoplastic lymphoid infiltrates were present in multiple tissues, primarily in the skin and subcutis. The results of a CBC from an apparently healthy male Steller's eider, at least 14 years old, also indicated this bird had an increased WBC count with a lymphocytosis. Serum submitted for protein electrophoresis indicated that this bird had a monoclonal gammopathy. Lymphocytes isolated from the male Steller's eider had low levels of proliferation in response to mitogens. The bird survived nearly 2 years without treatment, and the WBC count continued to increase during this time. The male Steller's eider maintained good body condition and exhibited normal behavior throughout the course of the disease until a sudden decline just prior to death. Lymphoma was diagnosed based on histopathology results. Infiltrates of atypical lymphocytes were observed in the bone marrow, proventriculus, ventriculus, heart, and eyes.

Contemporary diets of walruses in Bristol Bay, Alaska suggest temporal variability in benthic community structure.

BACKGROUND: Pacific walruses (Odobenus rosmarus divergens) are a conspicuous and important component of the Bristol Bay ecosystem and human social systems, but very little is known about walrus ecology in this region, principally their feeding ecology. The present work provides contemporary data on the diets of walruses at four haulout locations throughout Bristol Bay between 2014 and 2018. METHODS: We analyzed scat and gastrointestinal tract samples from these animals using quantitative polymerase chain reaction to amplify prey DNA, which allowed for diet estimates based on frequencies of prey item occurrence and on the relative importance of dietary items as determined from DNA threshold cycle scores. RESULTS: Diets were highly diverse at all locations, but with some variation in composition that may be related to the time of year that samples were collected (summer vs. autumn), or to spatial variability in the distribution of prey. Overall, polychaetes and tunicates had the highest frequencies of occurrence and relative abundances in 2014-15, but a major change in diet appears to have occurred by 2017-18. While some sample sizes were small, diets in these later years contrasted sharply, with a greater prevalence of sea cucumbers and mollusks, and reduced importance of decapods and fishes compared to the earlier years. Prey identified in scat samples from one collection site also contrasted sharply with those reported from the same location in 1981. The apparent temporal shifts in walrus prey may represent a changing benthic ecosystem due to warming waters in recent decades.

Monitoring nearshore ecosystem health using Pacific razor clams (Siliqua patula) as an indicator species.

An emerging approach to ecosystem monitoring involves the use of physiological biomarker analyses in combination with gene transcription assays. For the first time, we employed these tools to evaluate the Pacific razor clam (Siliqua patula), which is important both economically and ecologically, as a bioindicator species in the northeast Pacific. Our objectives were to (1) develop biomarker and gene transcription assays with which to monitor the health of the Pacific razor clam, (2) acquire baseline biomarker and gene transcription reference ranges for razor clams, (3) assess the relationship between physiological and gene transcription assays and (4) determine if site-level differences were present. Pacific razor clams were collected in July 2015 and 2016 at three sites within each of two national parks in southcentral Alaska. In addition to determining reference ranges, we found differences in biomarker assay and gene transcription results between parks and sites which indicate variation in both large-scale and local environmental conditions. Our intent is to employ these methods to evaluate Pacific razor clams as a bioindicator of nearshore ecosystem health. Links between the results of the biomarker and gene transcription assays were observed that support the applicability of both assays in ecosystem monitoring. However, we recognize the need for controlled studies to examine the range of responses in physiology and gene transcripts to different stressors.

Physiological and gene transcription assays to assess responses of mussels to environmental changes.

Coastal regions worldwide face increasing management concerns due to natural and anthropogenic forces that have the potential to significantly degrade nearshore marine resources. The goal of our study was to develop and test a monitoring strategy for nearshore marine ecosystems in remote areas that are not readily accessible for sampling. Mussel species have been used extensively to assess ecosystem vulnerability to multiple, interacting stressors. We sampled bay mussels (Mytilus trossulus) in 2015 and 2016 from six intertidal sites in Lake Clark and Katmai National Parks and Preserves, in south-central Alaska. Reference ranges for physiological assays and gene transcription were determined for use in future assessment efforts. Both techniques identified differences among sites, suggesting influences of both large-scale and local environmental factors and underscoring the value of this combined approach to ecosystem health monitoring.

Immune parameters in different age classes of captive male Steller's eiders (Polysticta stelleri).

The immune system is important for host defense against antigens, but little is known about Steller's eider (Polysticta stelleri) immunology. This study compared hematological parameters, serum protein levels, lymphocyte proliferation, heat shock protein levels and oxidative damage in four different age classes of captive male Steller's eiders. The hatch year cohort had significantly higher total white blood cell and lymphocyte counts. The second year cohort had significantly higher albumin, alpha globulins and lymphocyte proliferation, and significantly lower beta globulin levels. The 9 year old males had a significantly higher IgY:IgY(ΔFc) ratio. The oldest eiders in the study, 14 + year old males, had significantly higher serum IgY, pre-albumin and glutathione reductase activity, and the lowest lymphocyte proliferation. This study provided a baseline of immune parameters in captive male Steller's eiders, and the results suggested the parameters were influenced by age-related changes.

The physiological effects of oil, dispersant and dispersed oil on the bay mussel, Mytilus trossulus, in Arctic/Subarctic conditions.

Increasing oil development around Alaska and other Arctic regions elevates the risk for another oil spill. Dispersants are used to mitigate the impact of an oil spill by accelerating natural degradation processes, but the reduced hydrophobicity of dispersed oil may increase its bioavailability to marine organisms. There is limited research on the effect of dispersed oil on cold water species and ecosystems. Therefore, spiked exposure tests were conducted with bay mussels (Mytilus trossulus) in seawater with non-dispersed oil, Corexit 9500 and oil dispersed with different concentrations of Corexit 9500. After three weeks of exposure, acute and chronic physiological impacts were determined. The majority of physiological responses occurred during the first seven days of exposure, with mussels exhibiting significant cytochrome P450 activity, superoxide dismutase activity and heat shock protein levels. Mussels exposed to non-dispersed oil also experienced immune suppression, reduced transcription and higher levels of mortality. After 21 days, mussels in all treatments exhibited evidence of genetic damage, tissue loss and a continued stress response. Bay mussels are useful as indicators of ecosystem health and recovery, and this study was an important step in understanding how non-dispersed oil, dispersant and dispersed oil affect the physiology of this sentinel species in Arctic/subarctic conditions.

Phylogenetic and pathogenic characterization of novel adenoviruses isolated from long-tailed ducks (Clangula hyemalis).

Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections.

Pathogenesis of Streptococcus infantarius subspecies coli Isolated from Sea Otters with Infective Endocarditis.

The Gram positive bacterial coccus Streptococcus infantarius subspecies coli is increasingly linked with development of fatal vegetative infective endocarditis and septicemia in humans, sea otters (Enhydra lutris) and other animals. However, the pathogenesis of these infections is poorly understood. Using S. infantarius subsp. coli strains isolated from sea otters with infective endocarditis, this study evaluated adherence and invasion of epithelial and endothelial cells, adherence to extracellular matrix components, and macrophage survival. Significant adherence to endothelial-derived cells was observed for 62% of isolates, 24% adhered to epithelial cell lines, and 95% invaded one or both cell types in vitro. The importance of the hyaluronic acid capsule in host cell adherence and invasion was also evaluated. Capsule removal significantly reduced epithelial adherence and invasion for most S. infantarius subsp. coli isolates, suggesting that the capsule facilitates attachment to and invasion of epithelium. Enzyme-linked immunosorbent assay testing revealed that all isolates adhered significantly to the extracellular matrix components collagen IV, fibronectin, laminin and hyaluronic acid. Finally, significant bacterial survival following phagocytosis by macrophages was apparent for 81% of isolates at one or more time points. Taken collectively these findings indicate that S. infantarius subsp. coli has multiple pathogenic properties that may be important to host colonization, invasion and disease.

The influence of year, laying date, egg fertility and incubation, individual hen, hen age and mass and clutch size on maternal immunoglobulin Y concentration in captive Steller's and spectacled eider egg yolk.

Steller's eiders and spectacled eiders are sea duck species whose populations have declined significantly and infectious diseases could influence offspring survival. Therefore, the maternal transfer of immunoglobulin Y (IgY) into yolk was investigated in captive Steller's and spectacled eiders during the 2007-2013 breeding seasons. This project had two objectives: establish baseline IgY levels in Steller's and spectacled eider yolk under controlled captive conditions and evaluate the effect of year, laying date, egg fertility, egg incubation duration, individual hen, hen age and mass, and laying order to determine which variables influenced IgY levels. Average IgY concentrations were 0.03-0.48 mg ml(-1) in Steller's eider yolk and 0.10-0.51 mg ml(-1) in spectacled eider yolk. The year and individual hen influenced IgY concentration in Steller's and spectacled eider yolk. The laying date was negatively correlated with egg IgY levels for most Steller's eider hens, but laying order was positively correlated with egg IgY concentration for spectacled eiders.