Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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0005 Our understanding of the mode of action of the

enterotoxin at the molecular level has been greatly

facilitated by the use of in vitro model systems using

various cell lines. Vero (African green monkey

kidney) cells are particularly sensitive to the toxin.

Rat liver, HeLa, small intestinal epithelial cells, and

other cell lines have also been used as models.

0006 The enterotoxin causes rapid membrane bleb for-

mation in cultured Vero cells (Figure 3). Total inhib-

ition of amino acid transport and deoxyribonucleic

acid (DNA), ribonucleic acid (RNA), and protein

synthesis occurs within 30 min of exposure. Within

40 min, 75% of the Vero cells detach (from the plastic

culture flask) and nearly 50% are nonviable.

0007Disruption of the plasma membrane is the specific

action of the enterotoxin in Vero cells. Binding to the

cell membrane must occur and is an early event. This

does not occur in naturally resistant cell lines or in

cells selected for resistance to the toxin. A protein of

molecular weight 50 000 has been extracted from

rabbit intestinal brush border membranes. This

protein inhibits biological activity of enterotoxin in

Vero cells, suggesting that it has a role in enterotoxin

binding. No role has been found for ganglioside GM

(gangliosides constitute a family of acidic glycolipids

which are important membrane components) from

rabbit intestinal brush border membranes in binding

C. perfringens enterotoxin. This is in contrast to the

role ganglioside GM

plays in binding cholera toxin.

0008The enterotoxin causes functional holes of defined

size in Vero cell membranes. The result is an alter-

ation of membrane permeability, causing rapid water

and ion flux changes immediately after binding.

Osmotic stabilizers protect against such enterotoxin-

induced morphological and permeability changes,

further suggesting the membrane as the site of action.

With a net influx of water and ions, the membrane

stretches and bleb formation results. This is followed

by the loss of essential precursors through leakage.

With the interruption of macromolecular synthesis

the cell eventually dies. While such specific events in

an in vitro cell model cannot be directly extrapolated

to the effects of the enterotoxin in human intestinal

cells, such evidence is highly suggestive that the mem-

brane of the human small intestine is the primary site

of action of C. perfringens enterotoxin.

Treatment of Illness

0009As in most cases of enterotoxin-mediated human food

poisoning, symptoms are self-limiting. In the case of

C. perfringens, symptoms subside within 1 or 2 days.

Death is uncommon but has occurred in

institutionalized individuals, especially the elderly.

In such cases fluid and mineral replacement therapy

is essential.

Prevention and Control

0010The factors contributing to outbreaks of foodborne

illness by C. perfringens were identified long ago.

Those most frequently cited are: (1) preparation of

food too far in advance; (2) inadequate cooling; (3)

storage at ambient temperature; and (4) inadequate

reheating. Multiple factors are frequently cited, but

(a)

(b)

20 µm

fig0003 Figure 3 Effect of enterotoxin on morphology of Vero cells. (a)

Control culture showing typical Vero cell morphology. (b) Vero

cells after 30 min exposure to enterotoxin. Note the spherical

morphology and formation of blebs (arrows). Reprinted from

McClane B and McDonel J (1979) The effect of Clostridium perfrin-

gens enterotoxin on morphology, viability and macromolecular

synthesis in Vero cells. Journal of Cellular Physiology 99: 193, with

permission.

1406

CLOSTRIDIUM

/Food Poisoning by

Clostridium perfringens

failure to refrigerate properly large portions of

previously cooked foods, especially in institutional

settings, invariably summarizes the events leading to

an outbreak.

0011 Spores on raw meat and poultry can survive

cooking which effectively heat-activates the spores

to promote germination when the product reaches a

suitable temperature during cooling. Rapid, uniform

cooling is therefore imperative. Gravies, broth and

large pieces of meat should be cooled to 10



C within

2–3 h. The organism can multiply rapidly in the

slowly cooling masses of meat and poultry. Cooking

also drives off oxygen and thereby promotes anaer-

obic conditions; this is especially important in liquid

foods, and in rolled meats where the contaminated

outside surface is rolled into the middle. Cooked,

chilled foods should be reheated to a minimum

internal temperature of 75



C immediately before

serving, in order to destroy vegetative cells. As noted

in the preceding article, cooked meat should be kept

above 62.8



C or below 10



0012 Human carriers are not a danger. It is impossible to

prevent human carriers from handling food because

most people harbor C. perfringens in their intestinal

tract. Similarly, the organism is present in a wide

variety of foods. Hence preventive measures depend

largely on knowledge of proper food preparation and

storage techniques, especially temperature control.

Certain trade organizations related to the food service

industry offer short courses dealing with proper food-

handling procedures. Clearly, education of food

handlers is a key aspect in the prevention of human

food poisoning in general, and that caused by C.

perfringens in particular.

See also: Clostridium: Occurrence of Clostridium

perfringens; Detection of Clostridium perfringens;

Contamination of Food; Diarrheal (Diarrhoeal)

Diseases; Food Poisoning: Classification

Further Reading

Labbe

R (1989) Clostridium perfringens. In: Doyle M (ed.)

Foodborne Bacterial Pathogens, pp. 191–234. New

York: Marcel Dekker.

McClane B (2001) Clostridium perfringens. In: Doyle M,

Beuchat L and Montville T (eds) Food Microbiology:

Fundamentals and Frontiers, 2nd edn., pp. 351–372.

Washington, DC: American Society for Microbiology

Press.

Smith L and Williams B (1984) The Pathogenic Anaerobic

Bacteria, 3rd edn. Springfield, IL: Charles Thomas.

Stringer M (1985) Clostridium perfringens type A food

poisoning. In: Borriello S (ed.) Clostridia in Gastro-

intestinal Disease, pp. 117–141. Boca Raton, FL: CRC

Press.

Occurrence of

Clostridium

botulinum

J W Austin, Bureau of Microbial Hazards, Ottawa,

Canada

Introduction

0001Clostridium botulinum is a Gram-positive, anaer-

obic, rod-shaped, spore-forming bacterium that

produces the most potent biological toxin known,

botulinum neurotoxin. Strains of C. botulinum are

separated into seven types, A through G, based on

the serological specificity of the neurotoxin produced.

Human botulism, including foodborne, wound, and

infant botulism, is associated with types A, B, E, and,

very rarely, F. Types C and D cause botulism in

animals. To date, there is no direct evidence linking

type G to disease. Based on physiological differences

and 16s rRNA gene sequencing, the species is also

divided into four groups: (I) all type A and proteolytic

strains of types B and F; (II) all type E and nonpro-

teolytic strains of types B and F; (III) type C and D

strains; (IV) type G strains. The emphasis here will be

on groups I and II since they are involved in human

illness. This article will discuss the distribution of

botulinum spores in the environment and in foods,

the different factors affecting growth in foods, and

detection of the organism and its neurotoxin in foods.

Presence of

C. botulinum

in the

Environment

0002Spores of C. botulinum are commonly present in soils

and sediments, but their numbers and types vary,

depending on the location (Table 1). The possibility

of contamination of food with C. botulinum depends

on the distribution and incidence of spores in the en-

vironment where the agricultural products are grown.

0003C. botulinum spores are widely distributed in

North America, but the spore load varies consider-

ably, as does the predominating type. Soils in the USA

east of the rise of the Rocky Mountains usually con-

tain type B spores, while type A spores predominate

in the western USA. Most US type B strains are pro-

teolytic. Type C spores are localized in soils along the

Gulf of Mexico coast. Overall, type E is found infre-

quently, and only in damp to wet locations. However,

in the region around the Great Lakes, and particularly

around Green Bay of Lake Michigan, high numbers

of type E are found in shoreline and sediment samples.

C. botulinum type E is also found in the coastal areas

of the states of Washington and Alaska, and along the

CLOSTRIDIUM

/Occurrence of

Clostridium botulinum

1407

tbl0001 Table 1 Incidence of Clostridium botulinum in soils and sediments

Location Sample size (g) % Positive samples MPNper kg

Type (%)

ABC/DEF

Eastern USA, soil 10 19 21 12 64 12 12 0

Western USA, soil 10 29 33 62 16 14 8 0

New York – Florida coast 1 4 36 8 17 42 33 0

Florida–Texas coast 1 6 62 12 4 38 46 0

Great Lakes, sediment 1 2 24 0 0 0 100 0

Green Bay, Wisconsin, sediment 1 77 1280 0 0 0 100 0

California coast north of 36



N 5 16 29 5 5 0 90 0

California coast south of 36



N 5 10 20 44 50 0 0 6

Alaska, coast 1 49 660 0 0 0 100 0

Canada, St Lawrence River estuary,

sediment River

10 75 140 0 0 0 100 0

Canada, Gulf of St Lawrence, 10 8 8 0 0 0 100 0

Laurentian Trough, sediment Canada,

Gulf of St Lawrence,

10 19 21 0 0 0 100 0

Magdalen Shelf, sediment Britain, soil 50 10 2 0 100 0 0 0

Britain coast, sediment 2 4 18 0 100 0 0 0

Ireland, soil 50 18 4 0 100 0 0 0

Iceland soil 10 3 3 0 100 0 0 0

Faroe Islands, coast 10 1 1 0 0 0 100 0

Greenland, coast 10 37 46 0 0 0 100 0

Skagerrak, Kattegat sediment 6 100 >780 0 0 0 100 0

Finland

Fresh water 300 61 372 0 0 0 100 0

Baltic, offshore 500 88 1020 0 0 0 100 0

Baltic, coast 300 58 500 0 0 0 100 0

Trout farm, sediments 200 68 2020 0 0 0 100 0

Netherlands, soil 0.5 94 2500 0 22 46 32 0

Norway, coast 5 7 7 0 0 0 100 0

Scandinavian coast, sediment 6 100 >780 0 0 0 100 0

Sweden, miscellaneous soil 6 29 30 0 5 95 0

Sweden, inland 6 47 110 0 4 0 96 0

Sweden, coast 36 590 84 410 0 0 1 99 0

Danish, coast 10 67 110 0 0 0 100 0

Denmark, miscellaneous soil 10 13 15 0 93 7 0 0

Poland, Baltic coast 5 32 76 3 0 0 97 0

Czechoslovakia, soil 42 0 0 100 0 0

Switzerland, soil 12 44 48 28 83 6 0 27

Rome, Italy, soil 7.5 1 2 86 14 0 0 0

Bangladesh, sediment 10 37 19 0 0 100 0 0

China, Sinkiang, soil 10 70 25 000 47 32 19 2 0

Former United Soviet Socialist Republic, soil

North-eastern 2 0 14 0 86 0

South-eastern 13 8 17 0 73 0

Central 14 16 77 0 6 0

North-western 4 14 0 0 86 0

Far East 14 0 8 5 87 0

Indonesia, sediment 50 21 5 0 0 100 0 0

Iran, Caspian sea, sediment 2 17 93 0 8 0 92 0

Japan, Hokkaido, soil 5–10 4 4 0 0 0 100 0

Japan, Ishikawa, soil 40–50 56 16 0 0 100 0 0

Japan, Honshu, river sediment 100 73 18 0 10 12 78 0

Java, coast 10 13 14 0 33 67 0 0

Taiwan, soil 10 78 30 34 37 4 20 5

Thailand, sediment 10 3 3 0 0 83 17 0

Argentina, soil 34 67 20 0 0 5

Brazil, soil 5 35 86 57 7 29 0 7

Costa Rica, soil 1 23 260 14 14 72 0 0

Paraguay, soil 5 24 10 14 0 14 0 71

Kenya, soil 5 25 33 89 0 11 0 0

South Africa, soil 30 3 1 0 100 0 0 0

New Zealand, sediment 20 55 40 0 0 100 0 0

1408

CLOSTRIDIUM

/Occurrence of

Clostridium botulinum

Pacific, Arctic and Atlantic coastlines of Canada. The

distribution of types on the Pacific coast changes with

latitude; south of 36



N, the prevalent types shift from

E to A and B.

0004 Type B predominates in the terrestrial environ-

ments of the UK, Ireland, Iceland, Denmark, and

Switzerland. It is associated with most botulism out-

breaks in Spain, Portugal, Italy, France, Belgium,

Germany, Poland, the former Czechoslovakia,

Hungary, and the former Yugoslavia, indicating

wide distribution of this type in the European envir-

onment. Type B also predominates in the aquatic

environments of the UK. Most European type B

strains are nonproteolytic. The predominant serotype

from other aquatic environments is E. The highest

numbers of type E spores are found in Scandinavian

waters, particularly in the Sound and Kattegat,

between Denmark and Sweden. The highest overall

spore concentrations detected in Europe were

reported in The Netherlands, where a mixture of

types was found.

0005 Type E spores also predominate in most parts of the

former Soviet Union, except for the central portion,

where type B spores predominate. In general, surveys

of Asia report lower numbers, with the exceptions of

a high incidence of type E spores around the Caspian

Sea, and a high incidence of all types in the Sinkiang

district of China.

0006 Fewer surveys have been carried out in the south-

ern hemisphere. Spores of all types have been detected

in South America; type E has been found in fish,

oyster, and shrimp samples taken off the Brazilian

coast. Type A spores predominate in Brazilian and

Argentine soils. In the tropical regions of Asia, types

C and D replace type E as the predominant type in

aquatic environments.

0007 The distribution of spore types in fish and shellfish

is similar to that in the sediments from their respective

areas. Because the methods of sampling fish and shell-

fish differ widely, it is difficult to compare numbers.

0008 In summary, type A spores predominate in soils in

the western USA, China, Brazil, and Argentina, and

type B spores in the eastern USA, the UK, and much of

continental Europe. However, most US type B strains

are proteolytic, while most European strains are non-

proteolytic. Type E is the predominant type in north-

ern regions, and in most temperate aquatic regions

and their surroundings. Types C and D are found more

frequently in warmer environments. The reasons for

this distribution pattern are not well understood. Type

A appears to favor neutral to alkaline soils with low

organic content, consistent with its virtual absence in

the highly cultivated soils of the eastern USA and

Europe. Type E is psychrotolerant, which undoubt-

edly plays a role in its prevalence in the north and in

many aquatic environments. Similarly, the higher op-

timum growth temperature for types C and D is con-

sistent with their presence in warmer environments.

Presence of

C. botulinum

in Foods

0009The sequence of events resulting in foodborne botu-

lism begins when C. botulinum contaminates a food.

This often occurs during growth or harvesting, and is

most likely to occur when a product originates in an

environment with a high incidence of spores. How-

ever, contamination can also occur during or after

processing. There have been considerably fewer

surveys of foods for contamination with C. botuli-

num than environmental surveys, and they have

focused primarily on fish, meats, and infant foods,

particularly honey.

0010Fish may become contaminated with spores of C.

botulinum in their environment, or during processing

and handling. The presence of C. botulinum, mostly

type E, in fish is readily demonstrated, although the

incidence is lower than in environmental surveys.

The average level of contamination of prepared fish

in the USA appears to be higher than in Europe and

Asia. The highest contamination level reported was in

salted fish from the Caspian Sea.

0011The level of contamination of meats is generally

low. It appears lower in North America, where the

average most probable number (MPN) is about 0.1

spore kg

1

, than in Europe, where the average MPN

is about 2.5 spores kg

1

. The types most often associ-

ated with meats are A or B.

0012C. botulinum is common in soil and organic fertil-

izers. C. botulinum types A or B may be present on

fruits and vegetables, particularly those in close con-

tact with the soil. One product of particular concern

is cultivated mushrooms, in which up to 2.1 10

type B spores kg

1

have been detected.

0013Spores in honey and other infant foods pose a

unique hazard because, in some infants, the spores

are able to colonize the intestines, produce toxin, and

cause infant botulism. By 1984, honey had been

implicated as the likely source of botulinum spores

in 20 cases of infant botulism in California. Surveys

for the presence of C. botulinum spores in honey

Most probable number (MPN) calculated using the Halvorson-Zeigler equation log

(n/q), where n ¼ the total number of samples analyzed and q ¼ the

number of nontoxic samples.

As percentage of the types identified.

Adapted from Hauschild AHW (1989) Clostridium botulinum. In: Doyle MP (ed.) Foodborne Bacterial Pathogens, pp. 111–184. New York: Marcel Dekker.

CLOSTRIDIUM

/Occurrence of

Clostridium botulinum

1409

suggest that the botulinum spore level in random

samples of honey is in the order of 1–10 spores

1

. However, in honey samples associated with

infant botulism the level is approximately 10

spores

1

. Other infant foods have also been examined.

While C. botulinum has been detected in samples of

corn syrup and rice cereal, exposure of infants to

botulinum spores via these foods seems to be minimal

as the spore levels are low, and spores are unlikely to

multiply during manufacture and storage.

0014 Other foods examined, including dairy products,

vacuum-packed products, and convenience foods,

show a very low incidence of C. botulinum spores.

Factors Affecting Growth and Toxin

Production in Foods

0015 The main factors affecting growth of C. botulinum in

foods are temperature, pH, water activity (a

), redox

potential, added preservatives, and other microorgan-

isms. The growth of types A, B, E, and F, of groups I

and II, has been intensively studied. Traditionally,

food microbiologists have established maximum

and/or minimum limits for these parameters which

would permit growth of C. botulinum (Table 2), and

these limits have often been used in the control of C.

botulinum. However, these factors seldom function

independently; usually, they act in concert, often

having synergistic or additive effects. Models incorp-

orating pH, a

, and temperature are available to

predict the growth of C. botulinum under various

combinations of conditions.

Temperature

0016 Because foods are generally stored at low tempera-

tures, studies have focused on determining the

minimum temperatures permitting growth. The

established lower limits are 10



C for group I and

3.0



C for group II. However, these limits apply to

relatively few strains and depend on otherwise opti-

mum growth conditions. Production of toxin generally

requires several weeks at the lower temperature limits.

The optimum growth temperature is in the range 35–



C for group I organisms, and in the range 25–30



for those of group II. The upper temperature limits for

group I and group II organisms are approximately 45–



C and 40–45



C, respectively.

0017It is generally accepted that the minimum pH permit-

ting growth of C. botulinum group I is 4.6, and many

regulations worldwide use this limit. For group II, the

limit is about pH 5.0. The upper pH limits for growth

are in the pH range 8–9, but are of little practical

consequence. Many fruits and vegetables are suffi-

ciently acidic to inhibit C. botulinum by their pH

alone, while other products, such as marinated vege-

tables, are preserved by added acidulants. Several

factors influence the acid tolerance of C. botulinum,

including strain, substrate, temperature, nature of the

acidulant, the presence of preservatives, a

, and redox

potential. The growth of acid-tolerant microorgan-

isms such as yeasts and molds may raise the pH in

their immediate vicinity to a level that permits growth

of C. botulinum. C. botulinum can also grow in some

acidified foods if excessively slow pH equilibration

occurs. While high concentrations of proteins in la-

boratory media appear to protect C. botulinum and

permit growth at pH levels below 4.6, this does not

occur in foods preserved by acidity. Current regula-

tions stipulating a minimal acidity of pH 4.6 for the

control of C. botulinum are therefore valid.

Salt and

0018Salt (NaCl) is one of the most important factors con-

trolling C. botulinum in foods. Its inhibitory effect is

primarily due to the depression of a

and conse-

quently to its concentration in the aqueous phase,

also called the brine concentration (%brine ¼

NaCl 100/(%H

O þ%NaCl)). Under otherwise

optimal conditions, the growth-limiting brine con-

centrations are about 10% for strains of group I and

5% for strains of group II. These concentrations cor-

respond well to the limiting a

of 0.94 for group I and

0.97 for group II in foods where NaCl is the main a

depressant. The type of solute used to control a

may

influence these limits. Generally, NaCl, potassium

chloride, glucose, and sucrose show similar patterns,

while the use of glycerol reduces the growth-limiting

level by up to 0.03 units. The limiting a

may be

raised significantly by other factors, such as increased

acidity or preservatives.

Redox Potential

0019C. botulinum grows optimally at an oxidation–

reduction potential (E

)of350 mV, but growth

tbl0002 Table 2 Properties of group I and II Clostridium botulinum

Property Group I Group II

Toxin types A, B, F B, E, F

Minimum temperature for growth (



C) 10 3

Maximum temperature for growth (



C) 48 45

Minimum pH for growth 4.6 5

Inhibitory (NaCl) (%) 10 5

Minimum a

for growth 0.94 0.97

100



of spores (min) 25 <0.1

121



of spores (min) 0.1–0.2 <0.1

1410

CLOSTRIDIUM

/Occurrence of

Clostridium botulinum

initiation may occur in the E

range of þ30 to

þ250 mV. The presence of other inhibitory factors

lowers this upper limit. Once growth is initiated, the

declines rapidly.

0020 Modified atmosphere packaging is being used

increasingly to extend the shelf-life and improve the

quality of foods. Depending on the atmosphere and

the food, growth of C. botulinum may be inhibited or

stimulated. Many studies have shown that C. botuli-

num will grow as well in foods packed in air as in

vacuum-packed foods; the oxygen in the package

head space can be rapidly depleted by respiration of

background microorganisms or, in the case of modi-

fied-atmosphere packaging vegetables, by the plant

tissue itself. The safety of different atmospheres with

respect to C. botulinum should be carefully investi-

gated before use.

Preservatives

0021 Nitrite is important in producing the characteristic

color and flavor of cured food products, but its

most important role is the inhibition of C. botulinum.

It is more effective with decreasing pH, increasing

NaCl content, and addition of ascorbate or isoascor-

bate to the food product. Nitrite reacts with many

cellular constituents and appears to inhibit C. botuli-

num by more than one mechanism, one of which is

probably its reaction with essential iron-sulfur pro-

teins to inhibit the phosphoroclastic system which

supplies the cell with energy. The reactions of nitrite,

or nitric oxide, with secondary amines in meats to

produce nitrosamines, some of which are carcino-

genic, has led to regulations limiting the amount of

nitrite used.

0022 Other compounds which are active against C.

botulinum include sorbates, parabens, nisin and

other bacteriocins, phenolic antioxidants, polyphos-

phates, ascorbates, ethylenediaminetetraacetic acid

(EDTA), metabisulfite, n-monoalkyl maleates and

fumarates, and lactate salts. Extracts of certain

foods, including mace, bay leaf, and nutmeg extracts,

oils from garlic, onion, black pepper, clove, cinna-

mon, and origanum have all been shown to inhibit

the growth of C. botulinum. The use of natural or

liquid smoke has a significant inhibitory effect against

C. botulinum in hot smoked fish, reducing the inhibi-

tory water-phase NaCl concentration from 3.7% to

2.9–3.0%.

Other Microorganisms

0023 Other microorganisms have a significant role in the

control of C. botulinum in foods. Acid-tolerant yeasts

and molds may make the environment more favorable

for growth of C. botulinum. Other microorganisms

may inhibit C. botulinum, either by changing the

environment, or by producing specific inhibitory sub-

stances, or both. Lactic acid bacteria, including

Lactobacillus, Pediococcus,andStreptococcus spp.,

can inhibit growth of C. botulinum in meat products,

largely by reducing the pH, perhaps also by the pro-

duction of bacteriocins. Growth of Bacillus spp. that

produce antimicrobials has been shown to inhibit

growth of group II C. botulinum in foods. The use

of lactic acid bacteria and a fermentable carbohy-

drate, the ‘Wisconsin process,’ has been permitted

for producing bacon with a decreased level of nitrite

in the USA. The growth of other microorganisms may

also protect the consumer by causing spoilage that

would make a toxic product less likely to be con-

sumed.

Thermal Inactivation

0024C. botulinum spores of group I are very heat-resist-

ant. D values (the time required to inactivate 90% of

the population at a given temperature) vary consider-

ably among C. botulinum strains and depend on how

the spores are produced and treated, the heating

environment, and the recovery system. Spores of

types A and B are the most heat-resistant, having

121



values in the range 0.1–0.2 min. These spores

are of particular concern in the sterilization of canned

low-acid foods, and the canning industry has adopted

a D value of 0.2 min at 121



C as a standard for

calculating thermal processes. The z value is defined

as the temperature change necessary to bring about a

10-fold change in the D value. For the most resistant

strains, z values are approximately 10



C, which has

also been adopted as a standard. Actual z values may

vary by several degrees. Despite the variations in D

and z values, the adoption of a 12D process (‘botuli-

num cook’) as the minimum thermoprocess applied to

commercial canned low-acid foods by the canning

industry has insured the production of safe products.

The botulinum cook will produce a product that is

safe but not always sterile, depending upon the pres-

ence and levels of other, more heat-resistant, spores.

The thermal process of commercially canned foods

usually uses the more heat-resistant organisms, such

as Bacillus stearothermophilus, as target organisms

for the process.

0025Although strains of group 2 are considerably less

heat-resistant (D

100



< 0.1 min) than those of group

1, their survival in pasteurized, refrigerated products

is of concern because of their ability to grow at

refrigeration temperatures. D



values of type E in

neutral phosphate buffer are generally in the 0.2–

1.0 min range. While the D values are often higher

in foods, the z values are essentially the same.

CLOSTRIDIUM

/Occurrence of

Clostridium botulinum

1411

Inactivation by Irradiation

0026 C. botulinum spores are probably the most radiation-

resistant spores of public health concern. D values

(irradiation dose required to inactivate 90% of the

population) of group 1 strains at 50 to  10



C are

in the range 2.0–4.5 kGy in neutral buffers and in

foods. Spores of type E are only marginally more

sensitive, having D values in the range 1–2 kGy. The

goal of radappertization is to reduce the number of

viable spores of the most radiation-resistant C. botu-

linum by 12 log

cycles. D values are affected by

any pretreatment of spores, the presence of oxygen,

irradiation temperature, and irradiation and recovery

environments. Generally, spores are more sensitive in

the presence of oxygen or preservatives and at

temperatures above 20



Detection of

C. botulinum

and its Toxins in

Foods

0027 It is not necessary to isolate C. botulinum in pure

culture from foods in order to demonstrate its pres-

ence. Usually, the sample is inoculated into a non-

selective enrichment medium. If the neurotoxin is

present in the culture after incubation, the toxin-pro-

ducing organism must have been present originally.

0028 Botulinum neurotoxins are recognized by their

lethal action in mice and neutralization with specific

antisera. While several in vitro tests have been

developed, they are generally less sensitive and less

specific than the mouse bioassay which remains the

standard. The sample, or an extract prepared by

homogenizing it in a slightly acidic buffer, is clarified

by centrifugation and normally filter-sterilized. Tryp-

sin treatment may be required to activate low levels

of toxin from nonproteolytic strains. The prepared

sample is injected intraperitoneally into mice with

and without neutralization with antitoxin. Typical

signs of botulism are ruffled fur, pinched waist,

laboured breathing, limb paresis, and general paraly-

sis before death. Definitive results are obtained if

mice injected with untreated sample display symp-

toms and die within 72 h, while mice injected with

neutralized sample do not display symptoms and

survive.

0029 Common enrichment media for detecting viable C.

botulinum are cooked meat medium (CMM), CMM

glucose, chopped meat glucose starch (CMGS)

medium, and trypsin–peptone–glucose–yeast extract

(TPGY) broth, to which trypsin may be added

(TPGYT). Trypsin is necessary to activate the toxin

produced by group II organisms, and may also inacti-

vate potential inhibitors of C. botulinum such as

bacteriocins in mixed cultures. While foods may be

inoculated directly, the sediments of centrifuged

samples are preferred because potential growth

inhibitors are removed. At least two tubes of media

are inoculated. One is heated at 75–80



Cor60



depending on whether the suspected type belongs to

group I or II, to select for spores. Alternatively, spores

of group II may be selected by holding samples in

50% alcohol for 1 h before inoculation. The other

tube is incubated without any heating to allow devel-

opment of vegetative C. botulinum cells in case few or

no spores are present. Adding lysozyme to the

medium may increase recovery of heat-injured spores.

C. botulinum is identified after incubation of the

enrichment medium by toxin analysis of the super-

natant fluid, as outlined above.

See also: Canning: Principles; Chill Foods: Effect of

Modified-atmosphere Packaging on Food Quality; Chilled

Storage: Microbiological Considerations; Packaging

Under Vacuum; Use of Modified-atmosphere Packaging;

Fermented Foods: Origins and Applications;

Fermentations of the Far East; Heat Treatment: Chemical

and Microbiological Changes; Irradiation of Foods:

Basic Principles; Lactic Acid Bacteria; Microbiology:

Classification of Microorganisms; Detection of Foodborne

Pathogens and their Toxins; Preservatives:

Classifications and Properties; Food Uses; Water

Activity: Principles and Measurement

Further Reading

Austin JW, Dodds KL, Blanchfield B and Farber JM (1998)

Growth and toxin production by Clostridium botulinum

on inoculated fresh-cut packaged vegetables. Journal of

Food Protection 61: 324–328.

Collins MD and East AK (1998) Phylogeny and taxonomy of

the food-borne pathogen Clostridium botulinum and its

neurotoxins. Journal of Applied Microbiology 84: 5–17.

Daifas DP, Smith JP, Blanchfield B and Austin JW (1999)

Effect of pH and CO

on growth and toxin production

by Clostridium botulinum in English-style crumpets

packaged under modified atmospheres. Journal of

Food Protection 62: 1157–1161.

Daifas DP, Smith JP, Blanchfield B and Austin JW (1999)

Growth and toxin production by Clostridium botulinum

in English-style crumpets packaged under modified

atmospheres. Journal of Food Protection 62: 349–355.

Dodds KL and Austin JW (1997) Clostridium botulinum.

In: Doyle MP, Beuchat LR and Montville TJ (eds)

Food Microbiology. Fundamentals and Frontiers, pp.

288–304. Washington: ASM Press.

Graham AF, Mason DR, Maxwell FJ and Peck MW (1997)

Effect of pH and NaCl on growth from spores of non-

proteolytic Clostridium botulinum at chill temperature.

Letters in Applied Microbiology 24: 95–100.

Hatheway CL and Ferreira JL (1996) Detection and

identification of Clostridium botulinum neurotoxins.

1412

CLOSTRIDIUM

/Occurrence of

Clostridium botulinum

Advances in Experimental Medicine and Biology 391:

481–498.

Hauschild AHW (1989) Clostridium botulinum. In: Doyle

MP (ed.) Foodborne Bacterial Pathogens, pp. 111–184.

New York: Marcel Dekker.

Hauschild AHW and Dodds KL (1993) Clostridium botuli-

num. Ecology and Control in Foods. New York: Marcel

Dekker.

Hielm S, Hyytia E, Andersin AB and Korkeala H (1998) A

high prevalence of Clostridium botulinum type E in

Finnish freshwater and Baltic Sea sediment samples.

Journal of Applied Microbiology 84: 133–137.

Lyver A, Smith JP, Austin J and Blanchfield B (1998) Com-

petitive inhibition of Clostridium botulinum type E by

Bacillus species in a value-added seafood product

packaged under a modified atmosphere. Food Research

International 31: 311–319.

Lyver A, Smith JP, Nattress FM, Austin JW and Blanchfield

B (1998) Challenge studies with Clostridium botulinum

type E in a value-added surimi product stored under a

modified atmosphere. Journal of Food Safety 18: 1–23.

Roberts TA, Baird-Parker AC and Tompkin RB (eds) (1996)

International Commission on Microbiological Specifica-

tions for Foods: Microorganisms in Foods, vol. 5. Micro-

biological Specifications of Food Pathogens. London:

Blackie Academic and Professional.

Botulism

J W Austin, Bureau of Microbial Hazards, Ottawa,

Canada

Introduction

0001 Botulism is a neuroparalytic disease resulting from

neurotoxin-mediated inhibition of acetylcholine

release from peripheral nerve synapses, including

neuromuscular junctions. This results in a descending

symmetrical flaccid paralysis beginning with the

cranial nerves. Death may result from asphyxiation

caused by paralysis of the diaphragm. Botulinum

neurotoxin is produced by Clostridium botulinum

and, rarely, by C. butyricum or C. barati. Human

botulism is classified into four categories. Foodborne

botulism, the most common form worldwide, is

caused by ingestion of food contaminated with pre-

formed botulinum neurotoxin, usually type A, B, or

E. Infant botulism, first recognized in 1976 and now

the most common form of botulism in the USA, is

caused by ingestion of viable spores which colonize

the intestinal tract of infants less than 1 year of age

and produce toxin locally. The environment appears

to be the most common source of spores for infant

botulism, and honey is the only food which has been

associated with infant botulism. Would botulism is

due to infection of a wound with spores of C. botuli-

num which grow and produce toxin in situ. Wound

botulism associated with injection drug use has

increased dramatically since 1994. Most cases of

wound botulism occur among heroin users in Califor-

nia who inject black tar heroin subcutaneously. The

fourth category, adult infectious botulism, includes

adult cases with intestinal colonization and toxemia,

and resembles infant botulism. This entry will discuss

the epidemiology, clinical aspects, and prevention of

foodborne botulism, and give a brief overview of the

structure and mode of action of the neurotoxin.

Epidemiology

0002Since epidemiological data depend upon the reporting

systems, and the thoroughness of these systems varies

between countries, these data are scarce in large

areas of the world. Table 1 shows the data for coun-

tries with frequent outbreaks. Unrecognized and mis-

diagnosed cases of botulism do occur, as shown by a

1985 outbreak in Vancouver, Canada, where the ini-

tial diagnoses for 28 patients included psychiatric

illness, viral syndrome, laryngeal trauma, and overex-

ertion.

Incidence in North America

0003In Canada, since 1985, 4.4 outbreaks of foodborne

botulism occur annually, with an average of 2.75

cases per outbreak. Most botulism outbreaks in

Canada have occurred in northern and west-coast

native communities. The foods involved were mainly

raw meats from marine mammals, fermented meats

such as muktuk (meat, blubber, and skin of the beluga

whale), raw fish, or fermented salmon eggs. Type E

was implicated in almost every case. Toxin produc-

tion in raw meats can occur because the meats are

often held at ambient temperature for some time. The

problem with so-called fermented products is that the

level of fermentable carbohydrates is too low to

ensure a sufficiently rapid pH reduction to prevent

growth of C. botulinum. Commercial products have

been implicated in four incidents in Canada since

1971: bottled marinated mushrooms imported from

the USA; bottled garlic in oil, also imported from the

USA but temperature-abused locally; inhouse bottled

mushrooms; and temperature-abused pa

produced

in Quebec.

0004In the USA, 9.4 outbreaks of botulism occur annu-

ally, with an average of 2.5 cases per outbreak. Alaska

accounts for 16.2% of all the outbreaks in the USA

occurring from 1950 to 1996. All of the Alaskan

outbreaks have involved native Alaskans; they

CLOSTRIDIUM

/Botulism 1413

involved raw or fermented native foods, and type E

was usually implicated. In continental USA, the situ-

ation is quite different. Most of the implicated foods

were home-preserved vegetables. The relative preva-

lence of type A and type B outbreaks was about equal

in the east, but type A outbreaks were much more

prevalent in the west, where type A spores predomin-

ate in the environment. Most botulism outbreaks in

the USA are caused by home-processed foods. Com-

mercially processed foods account for only 7% of the

botulism cases occurring in the USA since 1970. Tem-

perature abuse of either food ingredients or the final

product was often the problem.

Incidence in Europe

0005 Poland reported by far the highest number of cases

annually. This probably reflects both a high local

incidence of botulism and a very thorough surveil-

lance system. In Poland and several other European

countries, including Italy, France, Spain, Germany,

Hungary, Portugal, Czechoslovakia, and Belgium,

the predominant type involved was B (Table 1). In

Poland, the use of weck jars (weckglas) to seal cooked

meats (mostly pork) hermetically under a vacuum led

to a significant number of botulism cases. Other

common vehicles of botulism in Poland include

sausages, ham, headcheese (brawn), liver sausage,

and bacon. The incidence of botulism was highest in

Poland during the period of social change in the

1980s, reaching a peak of 738 cases in 1982. The

number of botulism cases in Poland is seasonal, with

the greatest incidences in May to August and in De-

cember. This may correspond to increased slaughter-

ing of pigs in private farms and increased preparation

of home-preserved ‘wecked’ products. Commercial

foods, usually canned meat and fish, were responsible

for 26% of cases in Poland.

0006The foods most frequently implicated, except in

Italy and Spain, were home-preserved meats such as

ham, fermented sausages, or canned products. Of the

implicated foods in France, 12% were of commercial

origin, but the manufacturers were generally small

local establishments. In Italy and Spain, the most

commonly implicated foods were home-preserved

vegetables, most of which in Italy had been preserved

in oil or water; in Spain, all of the incriminated vege-

tables had been canned. Commercial products were

implicated in several outbreaks in Italy and included

canned tuna fish in oil, dairy products (mascarpone

cheese), low-acid canned vegetables, and pasteurized

eggplant in oil. Scandinavian countries recorded

fewer outbreaks, and these were mainly associated

with fish and type E. Type E botulism has been

reported in Greenland after consumption of raw seal

meat and raw seal intestines. An outbreak of type B

botulism, involving 12 cases, caused by commercial

salted raw pork occurred in Switzerland between 31

December 1993 and 12 January 1994. Botulism out-

breaks have been rare in the UK; however, a major

outbreak in 1989 involved 27 cases, with one death.

Type B toxin had been produced in a hazelnut pure

which was then used to flavor a yogurt produced by a

local dairy. Other European countries reported either

few or no botulism outbreaks.

Incidence in the Russian Federation

0007The Russian Federation recorded 1327 outbreaks

of botulism, involving 2300 cases and 182 deaths

from 1988 to 1992. Most of these outbreaks were

caused by type B, and most outbreaks were caused by

tbl0001 Table 1 Recorded outbreaks of foodborne botulism

Country Period No. of

outbreaks

No. of cases Average cases

peryear

Predominant

type

Predominant foodimplicated

Russia 1988 – 1992 1327 2300 460 B Home-preserved mushrooms, fish

Poland 1993 – 1997 432 565 113 B Home-preserved meat

China 1958 – 1989 745 2861 90 B Homemade fermented bean curds

Italy 1988 – 1998 412 37 B Homemade vegetable preserves in oil or water

USA 1950 – 1998 476 1134 23 A Vegetables

Hungary 1987 – 1996 117 191 19 B Meats

Germany 1988 – 1998 NA 177 16 B Meats

Argentina 1979 – 1997 277 15 A Vegetables

Japan 1951 – 1987 97 479 13 E Fish

Canada 1985 – 1999 66 179 12 E Fermented marine mammal meat

Spain 1988 – 1998 NA 92 8 B Home-preserved vegetables

France 1988 – 1997 93 72 7.2 B Home-cured ham

Portugal 1970 – 1984 13 50 3 B Home-smoked ham

Norway 1975 – 1997 10 22 1 E Rakfish – traditional fermented fish

Belgium 1988 – 1998 10 1 B Meats

NA, not available.

1414

CLOSTRIDIUM

/Botulism

home-preserved mushrooms. Other foods com-

monly involved in botulism outbreaks include fish

and pork. During this period, commercial foods, usu-

ally canned meats and vegetables, were implicated in

one to five outbreaks annually. Incidents associated

with fish occurred mainly in regions around the Black

Sea, the Sea of Azov, the Caspian Sea, and Lake

Baikal.

Incidence in Asia

0008 Only a few countries in Asia reported outbreaks of

foodborne botulism. Israel reported an outbreak in

1987 which affected eight people, with one fatality.

The incriminated food was kapchunka (salted, une-

viscerated whitefish) contaminated with type E toxin.

All the kapchunka consumed by the patients had been

purchased in New York City. While the high salt

concentration of the kapchunka would have pre-

vented growth of C. botulinum in the flesh of the

fish, C. botulinum likely grew in the intestines of the

fish and neurotoxin may have diffused into the flesh.

0009 Iran reported a high incidence of botulism. Of the

314 cases recorded between 1972 and 1974, 170,

comprising 63 outbreaks, were investigated. The ma-

jority of outbreaks (97%) were associated with type E

and with fish or fish products. However, fleshy por-

tions of fish were responsible for only 10% of the

outbreaks; the other 90% were caused by fish eggs.

These eggs (ashbal) are salt-cured for several months

and then eaten without further treatment. A large

outbreak, caused by locally made cheese stored

under oil and involving 27 cases with one fatality,

occurred in a northern district of Iran in 1997.

0010 China has recorded 745 botulism outbreaks from

1958 to 1989. Most were associated with type A,

followed by types B and E. The north-western pro-

vince of Xinjiang recorded the majority of outbreaks,

which were usually type A. Typically, the incrimin-

ated food was fermented bean curd. In the northern

provinces of Ningxia and Hebei, outbreaks associ-

ated with type B predominated. Type E predominated

in Xizang province. Neurotoxigenic C. butyricum

was the cause of an outbreak of foodborne type E

botulism in Guanyun, Jiangsu province, China, in

January 1994. Six cases of clinically diagnosed botu-

lism were reported after consumption of salted and

fermented paste made of soybeans and wax gourds.

Type E toxin was detected in the implicated food and

C. butyricum, but not C. botulinum, was isolated.

Subsequently, type E neurotoxin-producing C. butyr-

icum was isolated from soil from four sites in an area

near to the sites where the type E outbreak occurred.

C. butyricum has also been isolated from sevu (crisp

made of gram flour) implicated in a 34-case outbreak

of botulism in India in September 1996.

0011The majority of Japanese outbreaks occurred in

northern areas and were associated with type E and

fish or fish products. Izushi was the food most often

implicated. To prepare izushi, fleshy pieces of fish are

soaked in water for a few days, and then packed

tightly into a tub with cooked rice, vegetables, salt,

vinegar, and spices and left to ferment, often for

3 weeks or longer. Izushi is eaten without further

cooking. Two outbreaks in Japan were associated

with commercial food. A type A outbreak was caused

by vacuum-packaged, stuffed lotus rhizome and in-

volved 36 cases with 11 deaths. An outbreak caused

by imported bottled caviar with 21 cases and three

deaths was due to type B toxin. Taiwan has also

reported a few botulism outbreaks, but reports from

other Asian countries are rare.

Incidence in Other Areas

0012Argentina is the only country in the southern hemi-

sphere which reported a substantial number of

botulism outbreaks. These occurred mostly in the

provinces of Mendoza and Buenos Aires, and at a

latitude between 30



and 40



S. Most outbreaks were

associated with type A and the implicated foods were

usually vegetables. A recent outbreak of type A botu-

lism among bus drivers in Buenos Aires was caused by

consumption of matambre, a meat roll containing

vegetables and eggs. The matambre was wrapped in

a heat-shrunk plastic wrap and stored with inadequate

refrigeration. Mexico, Guatemala, Venezuela, Peru,

Brazil, and Chile have all reported few outbreaks.

0013Chad, Egypt, Kenya, Madagascar, and Zimbabwe

are the only African countries with confirmed out-

breaks. In April 1991, an outbreak with 91 reported

cases of type E botulism and 18 reported deaths oc-

curred in Egypt. The food implicated in this outbreak

was faseikh (uneviscerated, salted mullet fish) origin-

ating from a single shop in Cairo. In Kenya, two

outbreaks were caused by native foods: one by sour

milk prepared in a gourd, and one by consumption of

raw termites. One of the two outbreaks reported from

Madagascar was unusual; the numbers involved were

high, and type E toxin was associated with a meat

product. About 60 people were involved, with 30

deaths, and locally manufactured bologna was the

vehicle.

0014Since 1942, Australia has recorded only six out-

breaks of foodborne botulism, and none since 1991.

One outbreak of type A botulism was recorded in

New Zealand, caused by home-bottled fermented

mussels and watercress, a traditional Maori dish.

Unconfirmed Outbreaks

0015Several outbreaks of foodborne botulism have been

reported in the media and distributed over the

CLOSTRIDIUM

/Botulism 1415