Methods for detection of Clostridium botulinum toxin in foods

Botulism is a deadly disease caused by ingestion of the preformed neurotoxin produced from the anaerobic spore-forming bacteria Clostridium botulinum. Botulinum neurotoxins are the most poisonous toxins known and have been a concern in the food industry for a long time. Therefore, rapid identification of botulinum neurotoxin using molecular and biochemical techniques is an essential component in the establishment of coordinated laboratory response systems and is the focus of current research and development. Because of the extreme toxicity of botulinum neurotoxin, some confirmatory testing with the mouse bioassay is still necessary, but rapid methods capable of screening large numbers of samples are also needed. This review is focused on the development of several detection methods for botulinum neurotoxins in foods.     

Evaluation of the effect of acetylsalicylic acid on Clostridium botulinum growth and toxin production

The Republic of Georgia (ROG) has the highest incidence of botulism among all countries in the world, with most cases attributed to home-preserved vegetables. Based on epidemiologic data, the occurrence of botulism in ROG is lower in areas where aspirin (active ingredient, acetylsalicylic acid [ASA]) is added to home-canned vegetables. The objective of this study was to evaluate, with a broth medium, the antibotulinal activity of ASA to determine the possible role of ASA in preventing botulinum toxin production in home-canned vegetables. Trypticase-peptone-glucose-yeast (TPGY) broth (pH 7.0) with 0, 0.3, and 0.6 mg of ASA per ml was inoculated with a 10-strain mixture of proteolytic Clostridium botulinum type A and B spores at ca. 10(3) spores per ml. The inoculated broths were incubated at 31 degrees C under anaerobic conditions, and C. botulinum growth and botulinum toxin production were determined for up to 36 h. Results showed ASA in broth delayed (time to initial detectable toxin produced and amount of toxin produced), but did not prevent, both growth and toxin production by C. botulinum. These results would not provide a definitive explanation for differences in toxin production in canned vegetables prepared with and without aspirin.     

Probability of growth and toxin production by nonproteolytic Clostridium botulinum in rockfish stored under modified atmospheres

The potential risk of C. botulinum growth in fresh fish stored under modified atmospheres remains unclear. Few studies have identified qualitatively certain conditions leading to toxigenesis. This paper is the First of a series attempting to quantify the effect of a variety of parameters on the probability (P) of toxigenesis by one spore in fish. The factorial design experiments included red snapper tissue homogenate inoculated with a pool of nonproteolytic spores (5 type E, 4 type B and 4 type F strains) at 7 levels (10⁴−10⁻² per 3 g sample) and incubated at 4, 8, 12, 17 and 30°C under 3 modified atmospheres (vacuum, 100% CO₂ and 70% CO₂+30% air) for up to 21 days. At the 10⁰ spore/sample level the earliest time to detect toxin production at 4, 8, 12, 17 and 30°C under all modified atmospheres was > 21, 12, 9, 6 and 2 days, respectively. At the 10¹ spores/sample level the earliest times for the same temperatures were > 21, 9, 6, 3–6 and 1–2 days, respectively. The probability of toxigenesis was affected significantly (P < 0.005) by temperature, storage time, atmosphere×temperature, and temperature×time but not by atmosphere (P > 0.1). Using linear and logistic regression models, equations were derived which can predict the P of 1 spore initiating growth and toxigenesis by a particular day and at a particular temperature of storage. Studies involving other fish substrates are in progress.     

Prediction of toxin production by Clostridium botulinum in pasteurized pork slurry

A model pork slurry system was used to study factors controlling the growth of Clostridium botulinum types A & B (Roberts, Gibson & Robinson, 1981a,b). The following factors were studied in combination: sodium chloride (2.5, 3.5, 4.5% w/v on water); sodium nitrite (100, 200, 300 μg/g); sodium nitrate (0, 500 μg/g); sodium isoascorbate (0, 1000 μg/g); polyphosphate (Curaphos 700, 0, 0.3% w/v); heat treatment (none, 8°C/7 min, 80°C/7 min + 70°C/1 hr); at two pH levels and stored at: 15, 17.5, 20 or 35°C.
Analyses of results yielded a statistical model providing two formulae (for 'low' and 'high' pH slurries) which estimate the probability of toxin production in the pork slurry system within the limits defined above.     

Probability of growth and toxin production by nonproteolytic Clostridium botulinum in rockfish fillets stored under modified atmospheres

Whether toxin production by Clostridium botulinum precedes or follows spoilage of fish stored under modified atmospheres (MA), remains unclear. In this factorial design study we inoculated a pool of nonproteolytic C. botulinum spores (5 type E, 4 type B, and 4 type F strains) at 6 levels (10⁴ to 10⁻¹) between two rockfish fillets and then incubated the fillets at 4, 8, 12 and 30°C under vacuum, 100% CO₂ and 70% CO₂+30% air for 21 days. The probability of toxigenesis by one spore was significantly affected (P<0.005) by temperature (T) and storage time (S_t), and not (P>0.1) by MA, MA×T or MA×S_t. At the 10° spore/sample level, the earliest time to detect toxin production at 4,8,12 and 30°C under all MAs was >21, 15–21, 6–9 and 2 days, respectively. No toxin production was detected at 4°C. Only type B toxin was present in the toxic samples. At 30°C storage, spoilage of fillets followed toxigenesis. Using linear and logistic regression models, equations were derived that could estimate the lag phase and predict the probability of one spore initiating growth under a particular storage condition.     

Assay in mice for low levels of Clostridium botulinum toxin

When botulinum toxin at a low level such as 0.1 to 1.0 mouse intraperitoneal LD50 was injected subcutaneously into a mouse at the inguinocrual region, abdominal ptosis with local palsy developed. If this symptom is taken as a marker, 1.0 mouse intraperitoneal LD50 can be detected within 6 h and 0.1 LD50 within 24 h. The severity of symptoms and the time-to-death in days after injection of toxin were converted into scores to quantify the toxic activity. Over a wide range of dose, between 0.075 and 38.4 mouse intraperitoneal LD50, a linear relationship was obtained between the log dose and the score. By use of this method, low levels of toxin such as 0.1 mouse intraperitoneal LD50 can be titrated accurately and easily.     

Growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres.

To determine the safety of a high moisture bakery product, packaged under modified atmospheres, challenge studies were done on English-style crumpets (water activity [a(w)] 0.990, pH 6.5) inoculated postbaking with Clostridium botulinum types A and proteolytic B spores (5 X 10(2) spores/g). Products were packaged either in air, in air with an Ageless FX200 oxygen absorbent, or in a CO2/N2 (60:40) gas mixture, stored at ambient temperature (25 degrees C), and monitored for toxicity daily. All inoculated crumpets were toxic within 4 to 6 days and were organoleptically acceptable at the time of toxigenesis. Counts of C. botulinum increased to approximately 10(5) CFU/g at the time of toxicity. To determine the effect of baking on product safety, subsequent challenge studies were done on crumpets inoculated with 5 x 10(2) spores/g (baked weight basis) prior to baking. All crumpets were toxic after only 6 days, irrespective of packaging conditions, and toxigenesis again preceded spoilage. Temperature profile studies showed that the maximum internal temperature reached during baking was 97 degrees C, and the total baking process was equivalent to 0.03 min at 121 degrees C. The actual time to toxin production in both studies (4 to 6 days) correlated well with the predicted time (3.4 days) using the U.S. Department of Agriculture Pathogen Modeling Program (version 5.1) for proteolytic strains of C. botulinum. These studies confirm that high moisture bakery products, if contaminated with C. botulinum spores either pre- or postbaking, could pose a public health hazard, if packaged in air (in a high gas barrier package where O2 was depleted and CO2 was generated during storage) or under modified atmosphere packaging conditions and stored at ambient temperature.     

Growth and toxin production by Clostridium botulinum in steamed rice aseptically packed under modified atmosphere

Sales and consumption of ready-to-eat aseptic steamed rice products have increased manyfold in Japan over the past 10 years. To determine the safety of steamed rice (water content 60%, pH 6.5) aseptically packaged under modified atmosphere, challenge studies were performed using a mixture of Clostridium botulinum proteolytic strains (five strains of type A and five strains of type B). Atmospheric conditions of 0 and 15% oxygen (with 5% CO2 and 5% N2 as the balance) were used. No neurotoxins were detected, and organoleptically acceptable conditions persisted for 24 weeks at 15% oxygen conditions. However, botulinum neurotoxin was found in one of three samples at 12 weeks and in one of two samples at 24 weeks at 0% oxygen and 30 degrees C. When samples were inoculated with C. botulinum with amylase (0% oxygen), neurotoxin and sample spoilage was detected after only 1 week of storage. Challenge studies using proteolytic strains of C. botulinum mixed with Bacillus subtilis (amylase formers) also were performed with atmosphere conditions of oxygen at 0, 5, 10, and 15% (with 5% CO2 and 5% N2 as the balance). Under 10 and 15% oxygen conditions, neurotoxin was not detected after 1 week of storage, but sample spoilage was detected after the same period. Under 0% oxygen conditions, neurotoxin was detected at 1 week, but the sample remained organoleptically acceptable even after 2 weeks of storage. Both neurotoxin and sample spoilage were detected at 1 week of storage under 5% oxygen conditions. Based on these results, cocontamination of amylase-producing Bacillus with C. botulinum would increase the risk of foodborne botulism when aseptic rice samples are packed under low-oxygen conditions (<5%). Therefore, to ensure the safety of these products, packing under atmospheric containing more than 10% oxygen is recommended.     

Effect of pH and CO2 on growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres.

The effect of pH and CO2 on both growth of and toxin production by Clostridium botulinum in English-style crumpets, packaged under modified atmospheres was investigated using a 2 x 2 factorial experiment. English-style crumpets (water activity, 0.990; pH 6.5 and 8.3) were inoculated with C. botulinum spores types A and proteolytic B (500 spores/g), packaged in either 60% CO2 (balance N2) or 100% CO2, stored at ambient temperature (25 degrees C), and monitored daily for toxicity. Toxin was detected after 4 days in crumpets packaged in 60% CO2, irrespective of initial product pH. Toxin production was delayed 1.5 to 3 days in crumpets packaged under 100% CO2. Analysis of variance indicated a significant interaction effect of pH and %CO2 on time of earliest toxin detection. Delay of toxin production was greatest for high pH (8.3) crumpets. All products were organoleptically acceptable at the time of toxigenesis, and therefore, high moisture-high pH bakery products, if contaminated with spores of C. botulinum, could become hazardous if packaged in atmospheres containing CO2.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats

The effect of combinations of sodium chloride (2.5, 3.5, 4.5% w/v on water), sodium nitrite (100, 200, 300 μg/g), sodium nitrate (0, 500 μg/g), sodium isoascorbate (0,1000 μg/g, or equimolar with nitrite level) and polyphosphate (Curaphos 700; 0, 0.3% w/v), on the growth of Clostridium botulinum types A and B was studied in an experimental pork slurry system, without heating and after two heat treatments (80°C for 7 min and 80°C for 7 min plus 70°C for 1 hr) followed by storage at: 15, 17.5, 20 or 35°C for up to 6 months.
Statistical analyses showed that increasing salt or nitrite levels, adding isoascorbate or nitrate, using the highest heat treatment or decreasing the storage temperature all significantly reduced toxin production by Cl. botulinum . The addition of 0.3% polyphosphate (Curaphos 700) significantly increased toxin production. There were many significant two-factor interactions; the effect of increasing nitrite was relatively less if isoascorbate was present, at 4.5% salt, or at low storage temperature. The presence of isoascorbate also counteracted the increase in toxin production attributed to the presence of polyphosphate.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats V. Prediction of toxin production: Non-linear effects of storage temperature and salt concentration

While investigating the effects of potassium sorbate and pig breed, cut and batch of pork in a pork slurry system, non-linear effects of storage temperature and salt concentration on toxin production by Clostridium botulinum were detected. Predicted probabilities of toxin production after analysis by logistic regression, published previously, were re-examined and similar effects detected. Improved formulae for the probability of toxin production in a model pork slurry system are given and the implications of the non-linearity of storage temperature and salt concentration on the predicted probability of toxin production are discussed.     

阪崎肠杆菌-食品安全控制的新目标

自 196 1年英国首次报道阪崎肠杆菌引起婴儿脑膜炎病例以来 ,世界上相继有多个国家报道了新生儿阪崎肠杆菌感染事件 ,婴儿配方奶粉与疾病的暴发密切相关。在 2 0 0 3年第 35次食品卫生法典大会上 ,美国和加拿大提出了有关控制婴幼儿配方奶粉中阪崎肠杆菌的危险性框架 ,指出 1岁以下的易感婴幼儿感染阪崎肠杆菌后有生命危险。 2 0 0 4年第 36次食品卫生法典大会一致通过并设立了以加拿大为首的起草工作组 ,加速修订婴幼儿食品国际卫生操作规范 ,制定阪崎肠杆菌和其它可能导致婴幼儿健康危害的相关病原菌的微生物标准。     

Food safety panel sulphur dioxide in foods

The following are summaries of four of the papers presented at a symposium on ‘Sulphur dioxide in foods’, organised by the Food Safety Panel of the Food Group. It was held on 22 October 1980 at the Society of Chemical Industry, 14 Belgrave Square, London, SW1X 8PS. The papers so published are entirely the responsibility of the authors and in no way reflect the views of the Editorial Board of the Journal of the Science of Food and Agriculture.     

Risk of Clostridium botulinum type E toxin production in blue crab meat packaged in four commercial-type containers.

The aim of this investigation was to determine if a risk of Clostridium botulinum growth and toxin production existed in four different packaged crabmeat products. Freshly picked blue crab meat was inoculated with 10(3) to 10(4) spores per g of a mixed pool of four strains of C. botulinum type E (Beluga, Minnesota, G21-5, and 070). The lump crabmeat was packaged in four different packaging containers: (i) 12-oz copolymer polyethylene cups currently used by most crab processors; (ii) 12-oz copolymer polyethylene cups with heat-shrink, tamper-evident low-density polypropylene seals; (iii) 8-oz copolymer polyethylene cups with easy-open aluminum ends: and (iv) 8-oz copolymer polypropylene cups with integral tamper-evident pull-tabs. The packages were stored at either 4 degrees C for 21 days or 10 degrees C for 15 days. Storage at 10 degrees C was used to simulate temperature abuse. The mouse bioassay was used to detect the presence of C. botulinum toxin. Psychotrophic and anaerobic populations were enumerated and were found to increase with time regardless of packaging type. No botulinum toxin was detected in any of the four packaging types stored at 4 degrees C or 10 degrees C throughout the entire storage period.     

Clostridium botulinum spores and toxin in mascarpone cheese and other milk products.

A total of 1,017 mascarpone cheese samples, collected at retail, were analyzed for Clostridium botulinum spores and toxin, aerobic mesophilic spore counts, as well as pH, a(w) (water activity), and Eh (oxidation-reduction potential). In addition 260 samples from other dairy products were also analyzed for spores and botulinum toxin. Experiments were carried out on naturally and artificially contaminated mascarpone to investigate the influence of different temperature conditions on toxin production by C. botulinum. Three hundred and thirty-one samples (32.5%) of mascarpone were positive for botulinal spores, and 7 (0.8%) of the 878 samples produced at the plant involved in an outbreak of foodborne botulism also contained toxin type A. The chemical-physical parameters (pH, a(w), Eh) of all samples were compatible with C. botulinum growth and toxinogenesis. Of the other milk products, 2.7% were positive for C. botulinum spores. Growth and toxin formation occurred in naturally and experimentally contaminated mascarpone samples after 3 and 4 days of incubation at 28 degrees C, respectively.     

Bacteria associated with processed crawfish and potential toxin production by Clostridium botulinum type E in vacuum-packaged and aerobically packaged crawfish tails

Refrigerated vacuum-packaged storage has been shown to increase significantly the shelf life of fresh fish and seafood products, but the effect, if any, on the outgrowth and toxin production of Clostridium botulinum type E on cooked crawfish is unknown. Microflora associated with live crawfish reflect the microbial populations of the harvest water and sediments in which they are living. The presence or absence of specific pathogens in either vacuum-packaged or air-permeable bags of cooked crawfish have not been thoroughly evaluated. This study evaluates the potential survival and outgrowth of biological hazards in both vacuum-packaged and air-permeable-packaged cooked crawfish held at 4 and 10 degrees C for 30 days. During shelf-life studies of vacuum-packaged and air-permeable-bagged cooked crawfish, a total of 31 bacterial species were isolated and identified from crawfish samples using both selective and nonselective media. The only pathogens isolated from both vacuum-packed and air-permeable bags of processed crawfish samples during shelf-life studies were strains of Aeromonas hydrophila and Staphylococcus aureus. C. botulinum type E and Clostridium perfringens species were not isolated from any of the uninoculated crawfish samples. Cooked crawfish were inoculated with 10(3) C. botulinum type E spores per g of crawfish tail meat to determine whether cooked crawfish tails would support the growth of C. botulinum type E strains and produce toxin at refrigerated temperatures. Spore-inoculated crawfish tails were vacuum packaged in both a high barrier film and an air-permeable bag and stored at 4 degrees C and 10 degrees C for 30 days. C. botulinum toxin E was not detected in any of the spore-inoculated packages throughout the shelf-life study until day 30. Microbiological data from this study should be useful in the development and implementation of the hazard analysis and critical control point plans for processed crawfish tails.     

The stability of Clostridium botulinum type E toxin in salty and/or acid environment

The stability of preformed Clostridium botulinum type E toxin in sterile buffer-and salt-solutions and in some commercial fish products has been examined. It has been found that progenitor toxin is stable for weeks at room temperature in sterile culture filtrate, spoiling fish and in low acid fish products and that it is unaffected by sterile saturated salt (NaCl) solutions and in salted fish. In high acid feed fish (fish silage pH 2–4) some inconsistant increased toxin titres have been observed.
The activated toxin, on the other hand, decreased and increased in titre during several weeks of storage in culture filtrate with added trypsin. In sterile NaCl solutions the titre decreased by a factor of 10 to that of a progenitor toxin, but in spoiling raw and salted fish toxicity was lost when pH exceeded 7.5.
The public health significance of these results is discussed.     

Quantitative risk assessment for hazards that arise from non-proteolytic Clostridium botulinum in minimally processed chilled dairy-based foods

A modular process risk model has been constructed that describes the manufacture of dairy dessert products and hazards that arise from non-proteolytic Clostridium botulinum. The model describes batch manufacture and consumer storage of a family size generic dairy dessert but includes a realistic quantification that could apply to a specific food product. The dairy dessert sector is an expanding part of the UK market. The model includes modules that describe spore loads in raw materials, spore inactivation during thermal processing, volume partition and the population kinetics for non-proteolytic C. botulinum during sequential isothermal storage regimes. Where possible elements of uncertainty and variability are identified explicitly. The model is constructed as a belief network from published data and expert opinions. The model provides marginal probabilities, and associated sensitivities, for a range of endpoint measures centred on the toxicity of a single retail unit after an extended period of storage. The decimal reduction time for non-proteolytic C. botulinum spore populations at the highest (hold) temperature of the primary thermal process and the highest temperature experienced during poorly controlled (consumer) storage are dominant factors determining risks. Priorities for additional information to support risk assessments have been identified.