Identifying Behavioral Response Profiles of Two Common Larval Fish Models to a Salinity Gradient.

Salinization of aquatic systems is an emerging global issue projected to increase in magnitude, frequency, and duration with climate change and landscape modifications. To consider influences of salinity on locomotor activity of common fish models, we examined behavioral response profiles of two species, zebrafish (Danio rerio) and fathead minnow (Pimephales promelas), across a gradient of sodium chloride. Following each experiment, behavior was recorded with automated tracking software and then behavioral response variables, including locomotor (e.g., distance traveled, number of movements, duration of movements) and photolocomotor changes, were examined at several speed thresholds (bursting, cruising, freezing) to identify potential salinity responses. Zebrafish responses were significantly (p < 0.05) reduced at the highest treatment level (5.78 g/L) for multiple behavioral endpoints during both dark and light phases; however, fathead minnow responses were more variable and not consistently concentration dependent. Future efforts are needed to understand behavioral response profiles in combination with anthropogenic contaminants and natural toxins across the freshwater to marine continuum, considering salinization of inland waters, sea level rise, and transport of anthropogenic contaminants and algal toxins from inland waters to coastal systems.

Nutrients and salinity influence Prymnesium parvum (UTEX LB 2797) elicited sublethal toxicity in Pimephales promelas and Danio rerio.

The magnitude, frequency, and duration of harmful algal blooms (HABs) are increasing worldwide, primarily due to climate change and anthropogenic activities. Prymnesium parvum is a euryhaline and eurythermal HAB forming species that has expanded throughout North America, resulting in massive fish kills. Previous aquatic ecology and toxicology efforts supported an understanding of conditions resulting in P. parvum HABs and fish kills; however, the primary endpoint selected for these studies was acute mortality. Whether adverse sublethal responses to P. parvum occur in fish are largely unknown. To begin to address this question, molecular and biochemical oxidative stress (OS) biomarker responses and photomotor behavioral alterations were investigated in two common fish models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). Varying nutrient and salinity conditions influenced P. parvum related OS biomarkers and fish behavioral responses in zebrafish and fathead minnows, which were heightened by nonoptimal conditions for P. parvum growth. Such sublethal observations present important considerations for future aquatic assessments and management of P. parvum HABs.

Low dissolved oxygen increases uptake of a model calcium channel blocker and alters its effects on adult Pimephales promelas.

Human population growth accompanied with urbanization is urbanizing the water cycle in many regions. Urban watersheds, particularly with limited upstream dilution of effluent discharges, represent worst case scenarios for exposure to multiple environmental stressors, including down the drain chemicals (e.g., pharmaceuticals) and other stressors (e.g., dissolved oxygen (DO)). We recently identified the calcium channel blocker diltiazem (DZM) to accumulate in fish plasma exceeding human therapeutic doses (e.g., Cmin) in coastal estuaries impaired due to nonattainment of DO water quality standards. Thus, we examined whether DO influences DZM uptake by fish, and if changes in DO-dependent upatke alter fish physiological and biochemical responses. Low DO (3.0 mg DO/L) approximately doubled diltiazem uptake in adult fathead minnows relative to normoxic (8.2 mg DO/L) conditions and were associated with significant (p < 0.05) increases in fish ventilation rate at low DO levels. Decreased burst swim performance (Uburst) of adult fathead minnows were significantly (p < 0.05) altered by low versus normal DO levels. DO × DZM studies reduced Uburst by 13-31% from controls, though not significantly (p = 0.06). Physiological responses in fish exposed to DZM alone were minimal; however, in co-exposure with low DO, decreasing trends in Uburst appeared inversely related to plasma lactate levels. Such physiological responses to multiple stressors, when paired with internal tissue concentrations, identify the utility of employing biological read across approaches to identify adverse outcomes of heart medications and potentially other cardiotoxicants impacting fish cardiovascular function across DO gradients.

CRISPR-Generated Nrf2a Loss- and Gain-of-Function Mutants Facilitate Mechanistic Analysis of Chemical Oxidative Stress-Mediated Toxicity in Zebrafish.

The transcription factor Nrf2a induces a cellular antioxidant response and provides protection against chemical-induced oxidative stress, as well as playing a critical role in development and disease. Zebrafish are a powerful model to study the role of Nrf2a in these processes but have been limited by reliance on transient gene knockdown techniques or mutants with only partial functional alteration. We developed several lines of zebrafish carrying different null (loss of function, LOF) or hyperactive (gain of function, GOF) mutations to facilitate our understanding of the Nrf2a pathway in protecting against oxidative stress. The mutants confirmed Nrf2a dependence for induction of the antioxidant genes gclc, gstp, prdx1, and gpx1a and identified a role for Nrf2a in the baseline expression of these genes, as well as for sod1. Specifically, the 4-fold induction of gstp by tert-butyl hydroperoxide (tBHP) in wild type fish was abolished in LOF mutants. In addition, baseline gstp expression in GOF mutants increased by 12.6-fold and in LOF mutants was 0.8-fold relative to wild type. Nrf2a LOF mutants showed increased sensitivity to the acute toxicity of cumene hydroperoxide (CHP) and tBHP throughout the first 4 days of development. Conversely, GOF mutants were less sensitive to CHP toxicity during the first 4 days of development and were protected against the toxicity of both hydroperoxides after 4 dpf. Neither gain nor loss of Nrf2a modulated the toxicity of R-(-)-carvone (CAR), despite the ability of this compound to potently induce Nrf2a-dependent antioxidant genes. Similar to other species, GOF zebrafish mutants exhibited significant growth and survival defects. In summary, these new genetic tools can be used to facilitate the identification of downstream gene targets of Nrf2a, better define the role of Nrf2a in the toxicity of environmental chemicals, and further the study of diseases involving altered Nrf2a function.

Toward Less Hazardous Industrial Compounds: Coupling Quantum Mechanical Computations, Biomarker Responses, and Behavioral Profiles To Identify Bioactivity of SN2 Electrophiles in Alternative Vertebrate Models.

Sustainable molecular design of less hazardous chemicals promises to reduce risks to public health and the environment. Computational chemistry modeling coupled with alternative toxicology models (e.g., larval fish) present unique high-throughput opportunities to understand structural characteristics eliciting adverse outcomes. Numerous environmental contaminants with reactive properties can elicit oxidative stress, an important toxicological response associated with diverse adverse outcomes (i.e., cancer, diabetes, neurodegenerative disorders, etc.). We examined a common chemical mechanism (bimolecular nucleophilic substitution (SN2)) associated with oxidative stress using property-based computational modeling coupled with acute (mortality) and sublethal (glutathione, photomotor behavior) responses in the zebrafish (Danio rerio) and the fathead minnow (Pimephales promelas) models to identify whether relationships exist among biological responses and molecular attributes of industrial chemicals. Following standardized methods, embryonic zebrafish and larval fathead minnows were exposed separately to eight different SN2 compounds for 96 h. Acute and sublethal responses were compared to computationally derived in silico chemical descriptors. Specifically, frontier molecular orbital energies were significantly related to acute LC50 values and photomotor response (PMR) no observed effect concentrations (NOECs) in both fathead minnow and zebrafish. This reactivity index, LC50 values, and PMR NOECs were also significantly related to whole body glutathione (GSH) levels, suggesting that acute and chronic toxicity results from protein adduct formation for SN2 electrophiles. Shared refractory locomotor response patterns among study compounds and two alternative vertebrate models appear informative of electrophilic properties associated with oxidative stress for SN2 chemicals. Electrophilic parameters derived from frontier molecular orbitals were predictive of experimental in vivo acute and sublethal toxicity. These observations provide important implications for identifying and designing less hazardous industrial chemicals with reduced potential to elicit oxidative stress through bimolecular nucleophilic substitution.

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Comparative behavioral toxicology with two common larval fish models: Exploring relationships among modes of action and locomotor responses.

Behavioral responses inform toxicology studies by rapidly and sensitively detecting molecular initiation events that propagate to physiological changes in individuals. These behavioral responses can be unique to chemical specific mechanisms and modes of action (MOA) and thus present diagnostic utility. In an initial effort to explore the use of larval fish behavioral response patterns in screening environmental contaminants for toxicity and to identify behavioral responses associated with common chemical specific MOAs, we employed the two most common fish models, the zebrafish and the fathead minnow, to define toxicant induced swimming activity alterations during interchanging photoperiods. Though the fathead minnow (Pimephales promelas) is a common model for aquatic toxicology research and regulatory toxicology practice, this model has received little attention in behavioral studies compared to the zebrafish, a common biomedical model. We specifically compared behavioral responses among 7 different chemicals (1-heptanol, phenol, R-(-)-carvone, citalopram, diazinon, pentylenetetrazole (PTZ), and xylazine) that were selected and classified based on anticipated MOA (nonpolar narcosis, polar narcosis, electrophile, specific mechanism) according to traditional approaches to examine whether these comparative responses differ among chemicals with various structure-based predicted toxicity. Following standardized experimental guidelines, zebrafish embryos and fathead minnow larvae were exposed for 96 h to each compound then were observed using digital behavioral analysis. Behavioral observations included photomotor responses, distance traveled, and stimulatory, refractory and cruising locomotor activity. Though fathead minnow larvae displayed greater behavioral sensitivity to 1-heptanol, phenol and citalopram, zebrafish were more sensitive to diazinon and R-(-)-carvone. Both fish models were equally sensitive to xylazine and PTZ. Further, the pharmaceuticals citalopram and xylazine significantly affected behavior at therapeutic hazard values, and each of the seven chemicals elicited unique behavioral response profiles. Larval fish behaviors appear useful as early tier diagnostics to identify mechanisms and pathways associated with diverse biological activities for chemicals lacking mechanistic data.

Scalable process for mitigation of laser-damaged potassium dihydrogen phosphate crystal optic surfaces with removal of damaged antireflective coating.

We investigate an approach for the recycling of laser-damaged large-aperture deuterated potassium dihydrogen phosphate (DKDP) crystals used for optical switching (KDP) and for frequency conversion (DKDP) in megajoule-class high-power laser systems. The approach consists of micromachining the surface laser damage sites (mitigation), combined with multiple soaks and ultrasonication steps in a coating solvent to remove, synergistically, both the highly adherent machining debris and the laser-damage-affected antireflection coating. We identify features of the laser-damage-affected coating, such as the "solvent-persistent" coating and the "burned-in" coating, that are difficult to remove by conventional approaches without damaging the surface. We also provide a solution to the erosion problem identified in this work when colloidal coatings are processed during ultrasonication. Finally, we provide a proof of principle of the approach by testing the full process that includes laser damage mitigation of DKDP test parts, coat stripping, reapplication of a new antireflective coat, and a laser damage test demonstrating performance up to at least 12 J/cm2 at UV wavelengths, which is well above current requirements. This approach ultimately provides a potential path to a scalable recycling loop for the management of optics in large, high-power laser systems that can reduce cost and extend lifetime of highly valuable and difficult to grow large DKDP crystals.

Toward the Design of Less Hazardous Chemicals: Exploring Comparative Oxidative Stress in Two Common Animal Models.

Sustainable molecular design of less hazardous chemicals presents a potentially transformative approach to protect public health and the environment. Relationships between molecular descriptors and toxicity thresholds previously identified the octanol-water distribution coefficient, log D, and the HOMO-LUMO energy gap, ΔE, as two useful properties in the identification of reduced aquatic toxicity. To determine whether these two property-based guidelines are applicable to sublethal oxidative stress (OS) responses, two common aquatic in vivo models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), were employed to examine traditional biochemical biomarkers (lipid peroxidation, DNA damage, and total glutathione) and antioxidant gene activation following exposure to eight structurally diverse industrial chemicals (bisphenol A, cumene hydroperoxide, dinoseb, hydroquinone, indene, perfluorooctanoic acid, R-(-)-carvone, and tert-butyl hydroperoxide). Bisphenol A, cumene hydroperoxide, dinoseb, and hydroquinone were consistent inducers of OS. Glutathione was the most consistently affected biomarker, suggesting its utility as a sensitivity response to support the design of less hazardous chemicals. Antioxidant gene expression (changes in nrf2, gclc, gst, and sod) was most significantly (p < 0.05) altered by R-(-)-carvone, cumene hydroperoxide, and bisphenol A. Results from the present study indicate that metabolism of parent chemicals and the role of their metabolites in molecular initiating events should be considered during the design of less hazardous chemicals. Current empirical and computational findings identify the need for future derivation of sustainable molecular design guidelines for electrophilic reactive chemicals (e.g., SN2 nucleophilic substitution and Michael addition reactivity) to reduce OS related adverse outcomes in vivo.

Global Assessment of Bisphenol A in the Environment: Review and Analysis of Its Occurrence and Bioaccumulation.

Because bisphenol A (BPA) is a high production volume chemical, we examined over 500 peer-reviewed studies to understand its global distribution in effluent discharges, surface waters, sewage sludge, biosolids, sediments, soils, air, wildlife, and humans. Bisphenol A was largely reported from urban ecosystems in Asia, Europe, and North America; unfortunately, information was lacking from large geographic areas, megacities, and developing countries. When sufficient data were available, probabilistic hazard assessments were performed to understand global environmental quality concerns. Exceedances of Canadian Predicted No Effect Concentrations for aquatic life were >50% for effluents in Asia, Europe, and North America but as high as 80% for surface water reports from Asia. Similarly, maximum concentrations of BPA in sediments from Asia were higher than Europe. Concentrations of BPA in wildlife, mostly for fish, ranged from 0.2 to 13 000 ng/g. We observed 60% and 40% exceedences of median levels by the US Centers for Disease Control and Prevention's National Health and Nutrition Examination Survey in Europe and Asia, respectively. These findings highlight the utility of coordinating global sensing of environmental contaminants efforts through integration of environmental monitoring and specimen banking to identify regions for implementation of more robust environmental assessment and management programs.

High fluence laser damage precursors and their mitigation in fused silica.

The use of any optical material is limited at high fluences by laser-induced damage to optical surfaces. In many optical materials, the damage results from a series of sources which initiate at a large range of fluences and intensities. Much progress has been made recently eliminating silica surface damage due to fracture-related precursors at relatively low fluences (i.e., less than 10 J/cm(2), when damaged by 355 nm, 5 ns pulses). At higher fluence, most materials are limited by other classes of damage precursors which exhibit a strong threshold behavior and high areal density (>10(5) cm(-2)); we refer to these collectively as high fluence precursors. Here, we show that a variety of nominally transparent materials in trace quantities can act as surface damage precursors. We show that by minimizing the presence of precipitates during chemical processing, we can reduce damage density in silica at high fluence by more than 100 times while shifting the fluence onset of observable damage by about 7 J/cm(2). A better understanding of the complex chemistry and physics of cleaning, rinsing, and drying will likely lead to even further improvements in the damage performance of silica and potentially other optical materials.

Tissue-specific bioconcentration of the synthetic steroid hormone medroxyprogesterone acetate in the common carp (Cyprinus carpio).

The steroid hormone medroxyprogesterone acetate (MPA), commonly used in oral and injectable contraceptives, has been detected in surface and wastewaters near urban and agricultural areas in several rivers of the world. The objectives of this study were to examine the accumulative potential and tissue distribution of MPA in fish. A freshwater species, the common carp (Cyprinus carpio), was exposed to 100 µg/L of MPA for a 7-day period followed by a depuration phase in which fish were maintained in dechlorinated tap water for an additional 7 days. Tissues (muscle, brain, plasma, and liver) were sampled during the uptake (days 1, 3, and 7) and depuration (day 14) phases of the experiment. Tissue-specific bioconcentration factors (BCF) ranged from 4.3 to 37.8 and uptake was greatest in the liver>brain>plasma and lowest in the muscle. From a regulatory standpoint, MPA shows little tendency to bioaccumulate in fish.

Fracture-induced subbandgap absorption as a precursor to optical damage on fused silica surfaces.

The optical damage threshold of indentation-induced flaws on fused silica surfaces was explored. Mechanical flaws were characterized by laser damage testing, as well as by optical, secondary electron, and photoluminescence microscopy. Localized polishing, chemical leaching, and the control of indentation morphology were used to isolate the structural features that limit optical damage. A thin defect layer on fracture surfaces, including those smaller than the wavelength of visible light, was found to be the dominant source of laser damage initiation during illumination with 355 nm, 3 ns laser pulses. Little evidence was found that either displaced or densified material or fluence intensification plays a significant role in optical damage at fluences >35 J/cm(2). Elimination of the defect layer was shown to increase the overall damage performance of fused silica optics.

Risk of carcase contamination with Campylobacter in sheep sent for slaughter into an abattoir in Scotland.

Campylobacter species have been identified as the major cause of acute bacterial enteritis in the UK. However, the epidemiology of campylobacteriosis remains poorly understood. It has been suggested that the role of sheep in the epidemiology of Campylobacter has been underestimated. The objective of the present study was to assess the infection risk of Campylobacter in sheep meat as a potential risk for human campylobacteriosis and to establish any possible associations between the epidemiological factors considered in the study and the presence of Campylobacter on lamb carcases. The prevalence of Campylobacter obtained from faecal samples and swabs from fleeces and carcases was presented in a previous paper. Epidemiological data was collected through questionnaires in order to identify risk factors for the presence of Campylobacter on the carcases and to make recommendations, based on the results obtained, to prevent human campylobacteriosis.

Factors associated with cross-contamination of hides of Scottish cattle by Escherichia coli O157.

The putative source of hide contamination for 236 cattle in Scotland followed from the farm through to slaughter was determined using phage and verocytotoxin type data. The majority of cattle (84%) were found to have subtypes of Escherichia coli O157 on their hide that had not been found previously in any animal from the farm of origin, strongly suggesting that contamination occurred once animals had left the farm of origin. Using logistic regression analysis, several variables and factors were found to be strongly associated (P < 0.01) with cross-contamination of cattle hides at the univariate level; commercial transport to slaughter, transport with other animals, use of a crush, line automation, and increasing slaughterhouse throughput were all risk factors, while feeding hay in lairage, processing an animal earlier in a slaughter cohort, and cleaning the landing area poststunning were protective. In the multivariable model, with the slaughterhouse and the farm group included as random effects, factors associated with the cross-contamination of cattle hides were identified. Transport to the slaughterhouse by a commercial hauler had a borderline-significant association with increased odds of an animal having a cross-contaminated hide (odds ratio [OR] [95% confidence interval (CI)] = 5.7 [0.99, 33.0]; P = 0.05). At the slaughterhouse, providing hay to cattle waiting in lairage (OR [95% CI] = 0.04 [<0.01, 1.04]; P = 0.05) and cleaning the landing area (OR [95% CI] = 0.03 [<0.01, 1.15,]; P = 0.06) also had a borderline-significant association with decreased odds of an animal having a cross-contaminated hide. Although the prevalence of carcass contamination remains very low, targeted intervention at the preslaughter stage may have the potential to reduce further the risk to public health.

Risk factors for hide contamination of Scottish cattle at slaughter with Escherichia coli O157.

In the slaughter processing of cattle, contaminated hides have been identified as one of the major sources of Escherichia coli O157 carcase contamination. Logistic regression analysis was applied to data collected in a large scale study in Scotland involving 222 cattle forming 34 groups sent for slaughter from 30 farms to 10 slaughterhouses. Aspects of individual animal characteristics, farm management practices and slaughterhouse features were examined to identify potential risk factors for hide contamination at harvest. Two models were developed, the first in which slaughterhouse was modelled as a fixed effect, and a second model where slaughterhouse and farm groups were modelled as random effects. In the first model, there was a significantly increased risk of a carcase testing positive for E. coli O157 on the hide if either the hide of the carcase immediately before or after it on the line was contaminated (OR 3.6; 95% CI: 1.4-9.9). If both adjacent carcases had contaminated hides, the odds ratio for the study carcase having a contaminated hide rose to 11.5 (95% CI: 4.4-32.5). If animals were held in lairage, receiving hay as feed appeared to have a protective effect on hide contamination. Transportation to the slaughterhouse by haulier, as opposed to transport by the farmer, was associated with a 5.4 increase in the odds of E. coli O157 contamination. The use of a crush in the lairage, often employed when reading ear tags, was also found to significantly increase the odds of hide contamination with E. coli O157. In the second model, the inclusion of slaughterhouse and farm group as random effects resulted in two of the previously identified factors being associated with hide contamination. If at least one of the adjacent carcases on the line had a contaminated hide, the associated odds ratio was 6.6 (95% CI: 2.8-15.9), which rose to 22.7 (95% CI: 9.3-55.5) if both adjacent hides were contaminated. Receiving hay in lairage was found to be important to the model, although not significant in itself (OR 0.005; 95% CI: 1.2e(-6)-20.7). These results suggest that modifiable risk factors for hide contamination exist. However, in order best to reduce the prevalence of hide contamination at slaughter, individual slaughterhouse risk assessment and intervention strategies are appropriate.

Region-specific DNA methylation in the preimplantation embryo as a target for genomic plasticity.

It has been long known that the unique genetic sequence each embryo inherits is not the sole determinant of phenotype. However, only recently have epigenetic modifications to DNA been implicated in providing potential developmental plasticity to the embryonic and fetal genome, with environmental influences directly altering the epigenetic modifications that contribute to tissue-specific gene regulation. Most is known about the potential environmental regulation of DNA methylation, epigenetic addition of methyl groups to cytosine residues in DNA that acts in the long-term silencing of affected sequences. While most attention has been paid to the methylation of imprinted gene sequences, in terms of developmental plasticity there are many more parts of the genome that are methylated and that could be affected. This review explores the distribution of cytosine methylation in the genome and discusses the potential effects of regional plasticity on subsequent development. Widening our consideration of potentially plastic regions is likely to greatly enhance our understanding of how individuals are shaped not only by DNA sequence, but by the environment in which pluripotent embryonic cells are transformed into the many cell types of the body.

Spatial and temporal epidemiology of sporadic human cases of Escherichia coli O157 in Scotland, 1996-1999.

In Scotland, between 1995 and 2000 there were between 4 and 10 cases of illness per 100000 population per year identified as being caused by Escherichia coli O157, whereas in England and Wales there were between 1 and 2 cases per 100000 population per year. Within Scotland there is significant regional variation. A cluster of high rate areas was identified in the Northeast of Scotland and a cluster of low rate areas in central-west Scotland. Temporal trends follow a seasonal pattern whilst spatial effects appeared to be distant rather than local. The best-fit model identified a significant spatial trend with case rate increasing from West to East, and from South to North. No statistically significant spatial interaction term was found. In the models fitted, the cattle population density, the human population density, and the number of cattle per person were variously significant. The findings suggest that rural/urban exposures are important in sporadic infections.

Frozen storage of Escherichia coli O157 in buffered peptone water and its detection on bovine carcasses.

The adaptation of a standard Escherichia coli O157 isolation method involving immunomagnetic separation and a period of frozen storage was investigated. A series of experiments was designed to test the recovery of a bovine strain of E. coli O157 from buffered peptone water after a period of frozen storage at -80 degrees C. The effects of the addition of glycerol at 5 and 10%, freezing time, the number of freeze-thaw cycles, the method of freezing and the method of thawing, the inclusion of a resuscitation-and-incubation step, and the sensitivity of the isolation method were investigated. The most effective method of storing frozen samples for 6 months and recovering strains of E. coli O157 after storage was found to involve 6 h of incubation of sample material in buffered peptone water at 37 degrees C before frozen storage at -80 degrees C with 10% glycerol, a rapid thaw after frozen storage, and resuscitation at 27 degrees C for 1 h and incubation at 37 degrees C for 1 h to allow freeze-injured and stressed bacteria to recover with a period of growth prior to immunomagnetic separation isolation. There was no significant decrease in log counts of a bovine strain E. coli O157 over 6 months of frozen storage in buffered peptone water with 10% glycerol. With this method, it was possible to isolate E. coli O157 from naturally infected bovine carcasses after a period of frozen storage.