Integrating the Forensic Sciences in Wildlife Case Investigations: A Case Report of Pentobarbital and Phenytoin Toxicosis in a Bald Eagle (Haliaeetus leucocephalus).

The application of medical knowledge to the purpose of law is the foundation of forensic pathology. A forensic postmortem examination often involves the expertise of multiple scientific disciplines to reconstruct the full story surrounding the death of an animal. Wildlife poses additional challenges in forensic investigations due to little or no associated history, and the disruptive effects of decomposition. To illustrate the multidisciplinary nature of wildlife forensic medicine, the authors outline a case of secondary pentobarbital/phenytoin toxicosis in a bald eagle (Haliaeetus leucocephalus). The eagle was the single fatality in a group of 8 birds that fed on euthanized domestic cat remains that had been improperly disposed of in a landfill. Cooperation between responding law enforcement officers, pathologists, and other forensic scientists led to the successful diagnosis and resolution of the case.

Bacillus cereus in personal care products: risk to consumers.

Bacillus cereus is ubiquitous in nature and thus occurs naturally in a wide range of raw materials and foodstuffs. B. cereus spores are resistant to desiccation and heat and able to survive dry storage and cooking. Vegetative cells produce several toxins which on ingestion in sufficient numbers can cause vomiting and/or diarrhoea depending on the toxins produced. Gastrointestinal disease is commonly associated with reheated or inadequately cooked foods. In addition to being a rare cause of several acute infections (e.g. pneumonia and septicaemia), B. cereus can also cause localized infection of post-surgical or trauma wounds and is a rare but significant pathogen of the eye where it may result in severe endophthalmitis often leading to loss of vision. Key risk factors in such cases are trauma to the eye and retained contaminated intraocular foreign bodies. In addition, rare cases of B. cereus-associated keratitis (inflammation of the cornea) have been linked to contact lens use. Bacillus cereus is therefore a microbial contaminant that could adversely affect product safety of cosmetic and facial toiletries and pose a threat to the user if other key risk factors are also present. The infective dose in the human eye is unknown, but as few as 100 cfu has been reported to initiate infection in a susceptible animal model. However, we are not aware of any reports in the literature of B. cereus infections in any body site linked with use of personal care products. Low levels of B. cereus spores may on occasion be present in near-eye cosmetics, and these products have been used by consumers for many years. In addition, exposure to B. cereus is more likely to occur through other routes (e.g. dustborne contamination) due to its ubiquity and resistance properties of spores. The organism has been recovered from the eyes of healthy individuals. Therefore, although there may be a perceived hazard, the risk of severe eye infections as a consequence of exposure through contaminated near-eye cosmetics is judged to be vanishingly small. It is unlikely that more stringent microbiological standards for near-eye cosmetics will have any impact on the risk of severe eye infections caused by B. cereus, as these are not linked to use of personal care products.

Modelling bacterial growth of Listeria monocytogenes as a function of water activity, pH and temperature.

Temperature, pH and water activity are important factors controlling the microbiological safety of foods. To describe the growth rate of Listeria monocytogenes in relation to these factors, two equations have been developed. Both equations are based upon the Ratkowsky equation for temperature and growth rate. The first equation predicts growth rate at sub-optimal pH values, sub-optimal temperatures and sub-optimal water activities, the second model predicts growth throughout the entire pH range. The first model may be used to predict growth rates between pH 4.6-6.7, temperature range 5-35 degrees C and a water activity range of 0.95-0.997. The second model is valid throughout the pH range of 4.6-7.4 and the same temperature and water activity range as the first model.

An example of the stages in the development of a predictive mathematical model for microbial growth: the effects of NaCl, pH and temperature on the growth of Aeromonas hydrophila.

The stages involved in developing a predictive model are illustrated using data describing the effects of temperature (3-20 degrees C), NaCl concentration (0.5-4.5% w/v) and pH (4.6-7.0) on the aerobic growth of Aeromonas hydrophila (cocktail of 6 strains). Optical density measurements using micro-titre plates were used as an initial screen, to determine the appropriate sampling times for viable counts to be made and to determine the approximate boundaries for growth. Growth curves were generated from viable counts and fitted using a modified Gompertz equation. Quadratic response surface equations were fitted to the log of lag and generation times, in response to the variables of temperature, NaCl and pH (in terms of hydrogen ion concentration). The effects of various combinations of these controlling factors are described. Comparisons between predicted growth rates and lag times from our response surface equations and other models for growth of A. hydrophila, developed with viable count data and optical density measurements, are made, together with comparisons with data from the literature on the growth of this bacterium in foods.

Predicting microbial growth: graphical methods for comparing models.

Some simple computer-based graphics were used to compare different models predicting microbial growth responses of salmonellae to three factors (pH, sodium chloride concentration and incubation temperature). Simple linear regression, contour and three-dimensional surface plots all revealed gross differences between the predicted growth parameters from different growth models. Regression and contour plots were found to be more sensitive to small differences in surface topography, but three-dimensional surface plots provided a good overview.

The effects of temperature, pH, sodium chloride and sodium nitrite on the growth of Listeria monocytogenes.

An automated turbidimetric system using multiwelled plates was used to examine the effects of different combinations of NaCl (0.5-8.0% w/v), NaNO2 (0-400 micrograms/ml) pH (4.6-7.4) and temperature (5-30 degrees C) on the growth of Listeria monocytogenes in tryptone soya broth. The data presented clearly illustrate the combinations that permit visible growth of the organism. The ability of L. monocytogenes to grow at low pH levels was strongly influenced by incubation temperature as well as NaNO2 concentration. At 20 degrees C and below, no visible growth was detected, even with 50 micrograms/ml NaNO2 at pH 5.3 (or below) within 21 days. At pH 6.0 and above, NaNO2 had little effect in delaying visible growth except at higher concentrations and also at lower incubation temperatures.

A predictive model for the combined effect of pH, sodium chloride and storage temperature on the growth of Brochothrix thermosphacta.

Growth of Brochothrix thermosphacta was observed under ranges of pH (5.6-6.8), NaCl (0.5-8.0% w/v) and incubation temperature (1-30 degrees C). In order to compare different approaches, two models were used to fit growth curves to viable count data, and to calculate parameters from those fitted curves. Growth responses as a function of pH, NaCl and temperature were described with a quadratic function which was then used to predict growth within the limits where growth was observed. The predictions of the model show good agreement with published observations from other laboratories.

Predictive modelling of growth of Listeria monocytogenes. The effects on growth of NaCl, pH, storage temperature and NaNO2.

The effect of NaCl concentration (5.0 115.0 g/l). pH value (4.0-7.2), temperature (1-35 degrees C) and NaNO2 concentration (0 200 mg/l) on the growth responses of Listeria monocytogenes, in laboratory medium was investigated. The growth curves generated within this matrix of conditions were fitted using the function of Baranyi and Roberts (1994) and the growth responses modelled using a quadratic polynomial to produce response surfaces. Growth curves could then be regenerated for any set of conditions within the experimental matrix and values predicted for the growth rate, doubling time, lag time and time to 1000-fold increase. The model was validated using data from published literature and was found to give realistic predictions for doubling times in foods, including meat and meat products, milk, dairy products and vegetables. Predictions from this model (Baranyi and Roberts. 1994) compared favourably with those from the models of Buchanan and Phillips (1990), Murphy et al. (1996) and the Food MicroModel.

Development of thermal inactivation models for Salmonella enteritidis and Escherichia coli O157:H7 with temperature, pH and NaCl as controlling factors.

The thermal inactivation of Salmonella enteritidis phage type 4 and Escherichia coli O157:H7 as affected by temperature (54.5-64.5 degrees C), pH (4.2-9.6 with HCl or NaOH) and NaCl concentration (0.5-8.5% w/w) was studied. Cell suspensions in modified tryptone soya broth were heated in a submerged-coil heating apparatus and survivors were enumerated on tryptone soya agar incubated aerobically. For most thermal inactivation data there was a logarithmic decrease in the viable cell concentration over the initial 4-6 log10 reduction and D-values were fitted. In some cases, tailing of the survivor curves was observed with cells surviving longer than the D-values predicted. Models describing the effect of temperature, pH and NaCl concentration on the thermal inactivation of S. enteritidis and E. coli O157:H7 were produced. For both organisms, predicted z-values of 4.6-7.0 C degrees were obtained depending on conditions, with larger z-values at higher levels of NaCl. Optimum survival occurred between pH 5 and pH 7 and increasing acidity or alkalinity caused a decrease in the predicted D-values. At equivalent pH, acetic acid and lactic acid (at 0.5, 1 and 2% w/w) generally had a similar, or increased, lethal effect compared with HCl, whereas in most cases citric acid had a less lethal effect. For E. coli O157:H7, increasing NaCl concentration had a protective effect up to the maximum tested (8.5% w/w), while for S. enteritidis optimal survival at a NaCl concentration of 5-7% w/w was predicted. The models were validated in foods by comparing predictions with published data. Most (80%) of the predicted D-values from the S. enteritidis model were within the 95% confidence interval (within 2.45-fold of the published data) for different Salmonella serotypes in whole egg, egg albumen, egg yolk, beef and milk. Most (93%) of the predicted D-values from the E. coli O157:H7 model were larger than the limited published data for this organism in meat, poultry, milk and apple juice with 42% within the 95% confidence interval (within 2.05-fold of the published data). The D-value models were incorporated into Version 1, and subsequent versions, of the predictive microbiology software program, Food MicroModel.

The effect of growth temperature on the phospholipid and fatty acyl compositions of non-proteolytic Clostridium botulinum.

A non-proteolytic strain of Clostridium botulinum (NCIB 4270) was found to have a complex lipid composition, comprising five major phosphorus-containing lipids: phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylserine (PS) and a glycophospholipid of unknown structure (GPL), in order of abundance. Changing the growth temperature did not alter the lipid composition either qualitatively or quantitatively. The main fatty acyl components of the lipids are 14:0, 16:0 and 16:1. When the growth temperature was lowered from 37 to 8 degrees C, there was an increase in 14:0 from 16.4 to 37.5%, an increase in 16:1 from 10.5 to 22.5%, and a decrease in the proportion of 16:0 from 40.3 to 19.1%. There was also a decrease in the proportion of cyclopropane fatty acids (15:0cyc and 17:0cyc) from 7.3 to 0.5%, and in the equivalent chain length of the total fatty acids from 15.9 to 15.3 as the temperature was lowered. The same temperature-dependent changes occurred in the five major lipid classes examined. Despite reports of the presence of plasmalogenic forms of phospholipids (i.e. those lipids which have the acyl chain in the sn-1 position replaced by an alk-1-enyl group) in some Clostridium spp., none were detected in C. botulinum NCIB 4270 using either commercially available spray reagents or by gas-liquid chromatographic analysis of the products or acid methanolysis of total lipid extracts. It is concluded that non-proteolytic C. botulinum lacks plasmalogens, typical of other clostridia, in its membranes and instead modulates its fatty acid composition in response to temperature changes in a manner that is typical of other (non-clostridial) bacteria.

Modelling the growth, survival and death of microorganisms in foods: the UK food micromodel approach.

Techniques for the development of mathematical models in the area of predictive microbiology have greatly improved recently, allowing better and more accurate descriptions of microbial responses to particular environmental conditions, thus enabling predictions of those responses to be made with greater confidence. Recognising the potential value of applying these techniques in the food industry, the Ministry of Agriculture, Fisheries and Food (MAFF) initiated a nationally coordinated five-year programme of research into the growth and survival of microorganisms in foods, with the aim of developing a computerised Predictive Microbiology Database in the UK. This initiative has resulted in the systematic generation of data, through protocols which ensure consistency of methodology, so that data in the database are truly comparable and compatible, and lead to reliable predictive models. The approaches taken by scientists involved in this programme are described and the various stages in the development of mathematical models summarized. It is hoped that this initiative and others being developed in the USA, Australia, Canada and other countries, will encourage a more integrated approach to food safety which will influence all stages of food production and, eventually, result in the development of an International Predictive Microbiology Database.

Bacterial thermal death kinetics based on probability distributions: the heat destruction of Clostridium botulinum and Salmonella Bedford.

Despite the long history and excellent record of inactivation models used in thermal processing, there are relatively few approaches that attempt to describe the kinetics commonly observed. There are even fewer examples of models that allow the user to deal with the environmental conditions that influence these kinetics. We describe an approach that assumes a distribution of inactivation times within a population of bacterial cells. The concept allows for alternative interpretations of death kinetics and provides excellent descriptions of data generated with two important foodborne pathogens, Clostridium botulinum and Salmonella Bedford. The Salmonella Bedford data set used is unusual and perhaps unique in that it provides information where more than 50% of the population survival has been measured. These measurements are often overlooked or missed in experimental work but are essential when using a vitalistic approach, enabling calculation of a 50% lethal dose for destruction of bacteria. Use of the normal or Prentice distribution provided better fits to the data than other models commonly used to describe thermal death. There was no obvious bias in the fits even though significant tailing was evident. In addition, the procedure described allows data from all the conditions to be fitted rather than individual independent series. This enables a single equation to be derived that can be judged against the whole domain of the data. Approaches that provide accurate and unbiased descriptions of thermal death are likely to become increasingly important to ensure the safety of more marginal heat processes.

Quantitative microbiological risk assessment: principles applied to determining the comparative risk of salmonellosis from chicken products.

Ensuring microbiological safety requires identification of realistic hazards and the means of controlling them. The risk assessment framework proposed by Codex Alimentarius allows the impact of raw materials and processes to be appreciated, and the output can be used for risk management and communication. Mathematical models allow numerical information to be processed by a computer and interpreted to give quantitative or comparative risk assessments. In this example, models have been put together according to the Codex. Alimentarius principles, providing a quantitative risk assessment (QRA) of salmonellosis from frozen poultry products. This model-based QRA takes into account three types of information: occurrence and distribution of the agent, sensitivity of populations to infection (e.g., normal or susceptible), and the effect of cooking (in the factory or home) on concentration of the agent and hence risks of infection after product consumption. It only demonstrates the impact of a single-process step (heating) and the effect of changes in population sensitivity, raw material quality, and cooking regime on the final risk. The effects of growth and recontamination are not considered. To aid risk communication, the models have been visualized by means of displays and slider controls on a computer screen because effective communication is essential to encourage manufacturers and their product designers to assess the effect of changes in processing or materials on risk.

Survival of Escherichia coli in foods.

Studies describing the survival of Escherichia coli in foods, more often than not use the O157:H7 serovar as the target organism. Whilst E. coli O157:H7 is undoubtedly the predominant agent of concern for foodborne disease caused by enterohaemorrhagic E. coli (EHEC), a consequence of this concern is the commonly held view that this one serovar is 'atypical' in its response to stress conditions and therefore better able to survive adverse environments. Many of the studies published do not make comparisons with other E. coli (either commensal organisms or other pathogenic types) or other members of the Enterobacteriaceae, that would justify this view. Nevertheless, there has been a great deal of valuable data and information generated describing the fate of E. coli O157:H7 in a range of foods stored under various conditions. In many respects, the results of these studies are not surprising considering the survivability of other closely related pathogens, such as Shigella spp. This ability to survive in foods for long periods of time confirms the need for reliable control measures where contamination is possible or likely, e.g. proper handling and thorough cooking of beefburgers. The factors that may influence survival in different foods are described, with the intention of providing an insight in this area of food safety. Key considerations for carrying out survival studies are identified, with particular reference to methodologies used.

The effect of incubation time and temperature on growth of Escherichia coli on gradient plates containing sodium chloride and sodium nitrite.

Two-dimensional gradient plates are a convenient way of screening antimicrobial effects of preservative factors acting in combination across a broad range of physical and chemical conditions. We report the effects of sodium chloride, sodium nitrite and incubation temperature on the growth of Escherichia coli by staining, laser densitometry and computer graphics. Staining not only more easily distinguished the growth area but also gave an indication of the viability of cells present. 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride was the more useful of the two stains used. Inhibitory concentrations of sodium chloride decreased with reduced incubation temperature. The response of E. coli to combinations of salt and nitrite on gradient plates was very similar to its response in liquid medium.

Osmotic stress on dilution of acid injured Escherichia coli O157:H7.

To determine surviving numbers of Escherichia coli from cultures or food systems, dilution with 0.1% peptone is regularly used. Higher numbers of survivors could be obtained from an acid-treated culture if 0.5 mol l-1 sucrose was added to the 0.1% peptone. Sorbitol, glucose or sodium chloride, but not glycerol, could be used in place of sucrose. Using electron microscopy distinct differences could be seen between acid-treated and untreated cells. The osmolarity of the diluents ranged from 5 to 500 mosmol kg-1 H2O for the 0.5 mol l-1 sugar or glycerol solutions, to about 1000 mosmol kg-1 H2O for the salt solution. Maximum recovery diluent has an osmolarity of about 300 mosmol kg-1 H2O and resulted in recovery of similar numbers of injured cells as a 0.5 mol l-1 solution of sugar in 0.1% peptone. Taking into account the observed damage to acid-treated cells and the differences in osmolarity of the diluents, it is likely that dilution in 0.1% peptone imposed additional stress on the acid-injured cells which caused further cell damage. Dilution in a more osmotically stable solution alleviated this osmotic stress.

The application of a log-logistic model to describe the thermal inactivation of Clostridium botulinum 213B at temperatures below 121.1 degrees C.

In this work, the death of Clostridium botulinum 213B was measured at temperatures between 101 degrees C and 121 degrees C. It was found that at all temperatures tested, survivor curves deviated from log-linearity which prevented their description using traditional first order kinetics. The survivor curves were better described using a vitalistic approach and the log-logistic transformation proposed by Cole et al. (1993). A single equation was derived to describe all survivor curves over the temperature range tested and a comparison of predicted and measured data showed good correlation. The implications of the use of the vitalistic approach to the validity of the 'minimum botulinum cook' is discussed.

The germinability of spores of a psychrotolerant, non-proteolytic strain of Clostridium botulinum is influenced by their formation and storage temperature.

The formation and storage temperatures of Clostridium botulinum spores are shown to influence their subsequent ability to germinate. Spores were formed at 10 degrees, 20 degrees, 30 degrees and 37 degrees C and following harvest were stored as aqueous suspensions at 20 degrees C (ambient temperature), 4 degrees C (refrigerated) or -20 degrees C (frozen) for periods of up to 1 month. The spores formed at 20 degrees C germinated most rapidly and to the greatest extent. When the spores were germinated immediately after harvest (fresh), there was no difference in the germinability of those spores formed at 20 degrees or 30 degrees C, whether or not they had been heat-shocked before use. However, following storage overnight or longer, differences in the relative germinabilities of the different spore samples were seen. Spores which had been stored at ambient temperature overnight germinated significantly faster and to a greater extent than did those which had been stored for up to 1 month. Similar differences were also observed between spores germinated fresh and those stored overnight, when the spores were stored refrigerated or frozen. Germinability was also influenced by the temperature of storage, since there were differences between spores formed at the same temperature but stored at different temperatures for the same period of time: for example, when spores which had been formed at 20 degrees C were germinated at 10 degrees C following a heat-shock, those which had been stored at ambient temperature germinated faster and to a greater extent than did those which had been stored refrigerated or frozen. It is concluded that there is a complex interaction between formation, storage and germination temperatures, which determines spore germinability. The fact that the changes are time-dependent and can occur in the frozen state is taken to mean that they are physico-chemical rather than metabolic. It is also significant in relation to refrigerated foods which are at risk from Cl. botulinum in that changes which occur during cool or frozen storage can enhance the germinability of spores if the temperature rises above that of chill cabinets.

The effect of temperature on the germination of single spores of Clostridium botulinum 62A.

Phase-contrast microscopy coupled with image analysis has been used to study the germination of single spores of Clostridium botulinum and to investigate the variation of germination lag of individual spores in a population (biovariability). The experiment was repeated at five different temperatures between 20 degrees C and 37 degrees C to look at the effect of temperature on the biovaribility of the spore germination. Data analysis shows that the germination lag distribution is skewed, with a tail, and that its shape is affected by the temperature. The origin of this biovariability is not exactly known, but could be due to a distribution of characteristics (e.g. permeabilities) or molecules (e.g. lytic enzymes) in the spore population. The method developed in this study will help us to describe and better understand the kinetics of spore germination and how this is influence by different environmental factors such as temperature and other factors that influence germination.

Germination-induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores.

In this work, we have used spores of Bacillus subtilis that specifically induce bioluminescence upon initiation of germination as a rapid, real-time monitor of the effects of preservative treatments on germination. Using this tool, we have demonstrated that the combination of mild acidity (pH 5.5 to 5.0), lactic acid (0. 5%), and a pasteurization step (90 degrees C for 5 min) results in enhanced inhibition of spore germination compared with the effects of the individual treatments alone. Inhibition by the combination treatment occurred as a result of both direct but reversible inhibition, entirely dependent on the physical presence of the preservative factors, and permanent, nonreversible damage to the L-alanine germination apparatus of the spore. However, we were able to restore germination of the preservative-damaged spores unable to germinate on L-alanine by supplementing the medium with the nonnutrient germinant calcium dipicolinic acid. The demonstration that simple combinations of preservative factors inhibit spore germination indicates that food preservation systems providing ambient stability could be designed which do not adhere to the strict limits set by commonly accepted processes and which are based on precise understanding of their inhibitory action.