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

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antibodies, lipoproteins, some membrane proteins,

nucleic acids, and viruses.

0012 Agarose gels, although quite rigid, are much less

elastic than polyacrylamide and so are more easily

torn and require careful handling.

Polyacrylamide Gel

0013 Favored for producing well-defined, mechanically

stable, and chemically inert matrices of variable

pore size, polyacrylamide gel (PAG) is probably the

most widely used gel medium in electrophoretic food

analysis today. The gels are mechanically strong, easy

to handle, and completely transparent. Owing to the

relatively small pore size, diffusion is limited, so PAG

usually provides a better resolution for most proteins

(with a molecular weight less than 500 000) than

other gel media.

0014 PAG is formed through polymerization of acryla-

mide monomers with the cross-linking comonomer

N, N

-methylenebisacrylamide, in the presence of

free radicals usually provided by the chemical initi-

ator ammonium persulfate or the photochemical

initiator riboflavin. The polymerization reaction

is regulated through the addition of N, N, N

-tetramethylethylenediamine, which provides a

source of tertiary amines.

0015 The pore size of the gel can be easily adjusted by

controlling both the concentration of acrylamide

(% C) and the relative proportion of the cross-linking

agent BIS to acrylamide (% T). This results in well-

defined molecular sieving properties, providing an

additional separation effect dependent on molecular

size.

Sample Pretreatments

0016 If sample proteins are insoluble or liable to aggre-

gation or precipitation during an electrophoretic

separation, dissociating and unfolding agents such

as urea or nonionic detergents (e.g., octylglucoside

and Nonidet P-40) are added to the sample or to

the gel. Uncharged thiol reducing agents such as

b-mercaptoethanol or the odorless compounds

dithiothreitol or dithioerythritol are used to protect

free thiol groups in proteins against oxidation (loss

of enzymatic activity) and disulfide bond formation.

Indeed, since these reagents will also cleave disulfide

bonds, they can be added together with a dissoci-

ating agent such as urea or, preferably, sodium dode-

cyl sulfate (SDS) to cause the deliberate dissociation

of proteins and their aggregates into their constitu-

ent subunits. This approach is successfully used in

SDS–PAG electrophoresis where samples are run

with and without the use of these thiol reducing

agents to give an estimate of the degree of protein

cross-linking by inter-and/or intramolecular disulfide

bonds.

0017Prior to isoelectric focusing, samples must be

desalted, e.g., by ultrafiltration or dialysis.

Gel Electrophoresis

0018Gel electrophoresis is zone electrophoresis in a chem-

ically inert gel matrix, such as polyacrylamide or

agarose. The sample is applied in a small volume as

a narrow zone, e.g., in gel slots. As the electric field is

applied, each sample component migrates according

to its own mobility in a gel medium of constant pH

and ionic strength. The separation into ‘pure zones’ is

achieved by maximizing the differential rate of migra-

tion, while minimizing zone spreading (dispersion)

due to heat convection and diffusion.

0019At present, there are three different geometrical

forms in which gel electrophoresis can be carried

out: horizontal slabs, vertical slabs, or vertical cylin-

ders (rods) of gel, often also referred to as tube gels.

Slab gels in thinner layers are preferred to thicker slab

or tube gels, as they offer faster separation, sharper

zones, and more rapid and efficient cooling and sub-

sequent staining. In PAG electrophoresis, there is a

clear preference for the horizontal system utilizing

ultrathin gel layers polymerized on to carrier foils.

The advantages of horizontal versus vertical systems

are easier handling, use of premanufactured gels,

more efficient cooling, reduced material costs, avail-

ability of fully automated systems, and flexibility

towards other forms of electrophoresis such as iso-

electric focusing.

PAG Electrophoresis

0020PAG electrophoresis (PAGE) is the most widely used

method in the analysis of complex mixtures of

proteins. PAGE of proteins can be broadly classified

in the following way:

0021Homogeneous systems with only a single separat-

ing gel and using continuous buffer media.

0022Multiphasic (discontinuous) systems, where the

stacking gel, a nonrestrictive large pore gel, is

layered on top of the separating gel, a small-pore

gel. Each gel layer is made with different buffers,

which may vary in pH, and/or ion mobility, and/or

ionic strength (conductivity).

0023In the stacking gel of a multiphasic system, sample

components are separated in the isotachophoresis

mode with no molecular sieving effect. After passing

the boundary between stacking and separating gel,

the sample components are then separated by size

and charge in the usual way. The very high resolving

power of this method, which is often referred to as

ELECTROPHORESIS 2057

disc PAGE, is due to the formation of very sharp zones

produced by the gel and buffer discontinuities. The

production of thin starting zones makes disc PAGE

very suitable for use with dilute sample solutions.

0024 Gradient PAGE provides a resolution superior to

that of a gel of single concentration. Polyacrylamide

can be cast in slabs or tubes in which the concen-

tration of acrylamide increases in a continuous

manner (e.g., linear or concave) over the length of

the gel, thereby producing an increasing sieving effect

due to decreasing pore size.

0025 PAGE of food proteins is most efficient in the pres-

ence of the anionic detergent SDS. SDS wraps around

the polypeptide backbone, breaking up noncovalent

protein aggregates (dimers, tetramers, etc.), and

cancels differences in intrinsic charge of the proteins.

In this way, SDS converts the proteins to rods of

negative charges with equal charge densities, i.e., it

confers a negative charge to the polypeptide chains in

proportion to their length and imparts the same free

solution mobility to all proteins, regardless of their

identity. Separation of SDS-denatured proteins occurs

principally by sieving effects, with lower-molecular-

weight proteins migrating more rapidly through the

gel. The rate of migration of a protein can be com-

pared to the rate of migration of standard proteins of

defined molecular weight, and fairly accurate esti-

mates of protein molecular weight can be obtained.

In combination with pore size gradients and discon-

tinuous buffer systems, SDS–PAGE can be an even

more powerful and efficient method for protein

separation.

0026 Because SDS–PAGE separates molecules according

to size, it is a highly favored technique in the prepar-

ation of two-dimensional protein maps,a particularly

when coupled with a method separating princi-

pally according to charge differences in the second

dimension.

Isoelectric Focusing

0027 In isoelectric focusing (IEF), amphoteric analytes,

such as proteins and macropeptides, are separated

according to their isoelectric point (pI) values in a

stable and linear pH gradient. The gradient increases

from a low pH at the anodic end to a high pH at the

cathodic end. The samples may be applied at any

position, before or after the pH gradient has been

formed. When an electric field is applied, the charged

sample components move towards the electrode

of opposite charge. As each component migrates

through the pH gradient and progressively encoun-

ters differences in pH, it becomes increasingly less

charged. Eventually, it arrives at a pH equal to its

pI, where its net charge is zero, and its migration

stops. Thus, each component is concentrated, or

sharply focused, into a narrow zone at its pI position,

where it remains stationary as long as the electric field

is maintained. To counteract the decrease in solubility

of proteins approaching pH values close to their pI,

solubilizing agents such as urea may be included.

0028The pH gradient is most effectively formed with the

use of a complex mixture of synthetic amphoteric

molecules (aliphatic polyaminopolycarboxylic acids)

known as ampholytes (e.g., ampholines). When an

electric field is applied to these ampholytes, they

each migrate to their respective pI. A relatively stable

pH gradient can be formed over the range pH 2–11

and also almost any portion thereof. The develop-

ment of immobilized pH gradient gels (known as

immobilines) has led to a significant improvement.

In these gels, amino and carboxy groups (acrylamide

derivatives) are covalently attached to the gel-

forming polyacrylamide backbone.

0029IEF is performed in essentially nonsieving media,

such as in free solution with a density gradient, PAG

of high porosity or in agarose with very low electro-

endosmosis agarose. Horizontal flat-bed systems with

thin or ultrathin layers of PAG are the most suitable.

The commercial availability of premanufactured gel

slabs has made analytical PAGIEF in both ampholine

and immobiline systems even more efficient and ac-

curate. In addition, toxicity problems from handling

the neurotoxic acrylamide monomers are avoided. IEF

in agarose gels is particularly appropriate for large

molecules or when a second-dimension immunodiffu-

sion or immunoelectrophoresis stage is also to be used.

0030IEF is a powerful technique that can separate pro-

teins differing in pI by as little as 0001. IEF is also a

very simple and accurate method for determining the

pI of a protein.

Isotachophoresis

0031In isotachophoresis (ITP), sometimes also referred to

as steady-state stacking or displacement electrophor-

esis, separation results from the different mobilities of

identically charged ions in a discontinuous electrolyte

system with two different solutions, a leading and a

terminating electrolyte. The sample is inserted be-

tween these two electrolyte solutions. In a specific

separation, either cations or anions can be deter-

mined, but not both at once. If sample anions are

being determined, the leading electrolyte must con-

tain an anion of higher mobility (e.g., chloride) than

any of the sample anions of interest, whereas the

terminating electrolyte must contain an anion of

lower mobility (e.g., glycine) than any of the sample

anions of interest. When the electric field is applied,

all negatively charged ions begin to move towards the

anode, arranging themselves in order of mobility.

2058 ELECTROPHORESIS

Separation of the sample ions takes place between

the leading and terminating electrolytes during

migration. When the system has reached equilibrium,

each individual ionic sample component moves sep-

arately as a ‘pure’ band. Each pure band is stacked

between the sample component of next highest and

lowest mobility. Thus, in the ‘anion-train,’ consecu-

tive sample analyte zones form behind the leading ion

zone (chloride), which acts as the ‘locomotive,’ and in

front of the terminating ion zone (glycine). The

sample zones travel in order of decreasing mobility

at the same velocity as the leading electrolyte zone.

These differences in mobility cause a stepwise change

in the electric field strength from one analyte zone to

the next, resulting in an important concentrating

effect. Backward or forward diffusion into a preced-

ing or following band is not possible. As a conse-

quence, the concentrating or stacking effect at the

boundary between bands produces sharp zones and

prevents diffusional broadening.

0032 Analytical-scale ITP is routinely performed in free

solution in small-bore capillary tubes and with the use

of a constant current at a well-defined pH value, and

with on-line detection. The resolved bands emerge as

rectangular peaks, not as the common Gaussian-

shaped curves in chromatographic separations.

Bands can be identified by measuring the electric

field strength within the band, by placing two micro-

electrodes side by side in the capillary channel. Quan-

titative information is contained in the length of the

band. In ITP, electrical detection is most commonly

used, although thermal and ultraviolet absorption

detection are also applied.

Analysis of Separated Zones

0033 The most important techniques for analysis of elec-

trophoretically separated proteins are summarized in

Table 2. Other techniques such as autoradiography

and fluorography of radiolabeled analytes so far have

not played a significant role in food analysis.

Staining

0034 The most common procedure for analysing proteins

separated in gels is staining with a variety of dyes that

bind to proteins. Usually, the staining procedure in-

volves the use of a fixative, such as trichloroacetic

acid, to precipitate proteins and prevent their diffu-

sion out of the gel. Gels are then soaked in a staining

solution, such that the entire gel is uniformly stained.

Nonprotein-bound dye must be removed in a destain-

ing rinse. The length of time required for both the

staining and destaining steps primarily depends on

the thickness of the gel, with the use of ultrathin gel

slabs saving a considerable amount of time.

0035Proteins in gels are most frequently stained with

Coomassie Blue or Amido Black dyes, or with photo-

graphic amplification systems using silver. The detec-

tion limits for Coomassie Blue staining are roughly

100 ng of protein in a band, whereas silver staining is

about 100 times more sensitive. Once the gel is

stained, it can be photographed or scanned by densi-

tometry for a record of the position and intensity of

each band. In general, the accuracy and precision

obtainable from this quantitation procedure are not

on a par with what is routinely obtained by high-

performance liquidchromatography.(See Chromatog-

raphy: High-performance Liquid Chromatography.)

0036Specific staining techniques exist for phosphopro-

teins, lipoproteins, glycoproteins, and nucleic acids.

Enzymes can be localized by assaying for their enzym-

atic activity, e.g., by converting substrates to spar-

ingly soluble products, which can then be coupled

chemically to azo dyes. Agarose gels and cellulose

acetate strips are very suitable for direct localization

of immunoreagents due to their pore size. Immuno-

fixation and immunoprinting are popular techniques

where antibodies and antigens, respectively, migrate

towards each other by diffusion (immunodiffusion).

Subsequent staining after the removal of unbound

antigens and antibodies allows selective detection of

specific protein fractions.

Blotting

0037Blotting refers to the transfer of separated zones of

macromolecules, e.g., proteins or nucleic acids, to

thin sheets of derivatized paper or adsorptive mem-

brane matrix, such as nitrocellulose or polyvinyldi-

fluoride (PVDF) to which they bind. Immobilized on

the surface of a thin matrix, macromolecules are more

easily and uniformly accessible to detection reagents

and react more sensitively and faster. The common

transfer methods are based on simple diffusion (diffu-

sion blotting), solvent flow with/without vacuum

(capillary or Southern blotting), or electrophoretic

elution (electroblotting). The detection of transferred

protein components on blots is carried out with all

the procedures described above, specific assays for

enzymes included.

Immunoblotting/Enzyme Labeling

0038Immunological detection of proteins is suitable with

all types of immobilizing transfer matrix. After pro-

teins or nucleic acids are transferred, all additional

matrix binding sites must be blocked with an excess

of nonspecific protein. Then, an antibody specific

for a certain class or type of protein is bound and,

finally, a second antibody directed against the first

antibody. This second antibody can be fluorescence-

labeled, radiolabelled, or conjugated to an enzyme for

ELECTROPHORESIS 2059

location by ultraviolet illumination, autoradiog-

raphy, or enzyme activity, respectively. Almost any

enzyme for which a suitable assay method exists

could be coupled in theory to an antibody by cross-

linking reagents (e.g., carbodiimide, glutaraldehyde,

etc.), but alkaline phosphatase and horseradish per-

oxidase, for which there are several simple and sensi-

tive detection methods, are very popular. The use of

these enzyme-labeled antibodies in combination with

blotting results in extremely sensitive procedures,

which can clearly detect as little as 100 pg of protein

on a membrane. (See Enzymes: Uses in Analysis.)

Immunoelectrophoretic Techniques

0039 Immunoelectrophoresis consists of a combination of

an electrophoretic step with the subsequent precipita-

tion of antigen–antibody complexes (immunoprecipi-

tates). The most common procedures in food analysis

rely upon the migration of antigenic proteins through

or into an antibody-containing gel. Buffers and pH

values are usually chosen so that only the antigens

migrate, and the antibodies either do not move at all

or, at most, migrate only very slowly and thus remain

evenly distributed throughout the gel during the whole

electrophoresis. The most popular techniques are:

0040 Counterimmunoelectrophoresis, sometimes re-

ferred to as countercurrent or crossed-over im-

munoelectrophoresis: in agarose gel of high

electroendosmotic flow, antigens electrophoreti-

cally move towards uncharged antibodies, which

are carried by the electroendosmotic flow in coun-

tercurrent, and precipitin arcs are formed.

0041 Grabar/Williams method: normal zone electro-

phoresis in agarose gel is followed by an immuno-

diffusion stage. Antibody diffusion from troughs

in the gel cut parallel to the line of electrophoreti-

cally separated components and precipitin arcs are

formed.

0042 Laurell rocket techniques: antigen proteins are

transported electrophoretically through a gel slab

containing antibodies at a pH value at which the

antibodies remain essentially immobile, resulting

in the formation of ‘rocket’-shaped precipitate

peaks whose height and area are linearly related

to the antigen concentration. These techniques

allow quantitative analysis of antigens, but are

not applicable to complex mixtures.

0043 Crossed immunoelectrophoresis. Agarose zone

electrophoresis or isoelectric focusing in one

dimension is followed by electrophoresis into an

antibody-containing gel slab in the second dimen-

sion, with the formation of mountain-like precipi-

tation peaks. Qualitative and/or quantitative

analysis of peak area is possible.

0044For many purposes, the formation of an opaque

precipitation are in a transparent gel will be relatively

clear and perfectly adequate. If desired, the sensitivity

of detection can be greatly enhanced by the applica-

tion of the usual protein-staining techniques (see

above) after the removal of nonprecipitated protein

(uncomplexed antigens and antibodies). An alterna-

tive to staining is the use of radiolabeled, fluorescent,

or enzyme-tagged reactants (see Immunoblotting/

Enzyme Labeling).

0045The use of immunoelectrophoresis in food analysis

is limited by the availability of specific antibodies, so

that cross-reactions with other nontargeted proteins

are avoided. (See Immunoassays: Principles.)

Capillary Zone Electrophoresis

0046Although conventional gel electrophoresis is ex-

tremely powerful and versatile, it is often a slow,

labor-intensive technique. Analyses often require

several hours, and often several days to complete.

But in the last 10 years, rapid instrumental methods

have been developed for free solution electrophoresis

in capillary tubes with on-line detection. Existing new

techniques currently available to the analytical chem-

ist are capillary electrophoresis (CE) in general, and

capillary zone electrophoresis (CZE) in particular.

0047CZE is performed as free solution electrophoresis in

capillaries with inside diameters of only 20–200 mm.

These small dimensions aid heat dissipation and

minimize zone broadening caused by molecular diffu-

sion and convection currents in the electrophoretic

medium. As a consequence, relatively high voltages

can be used, which allow highly efficient separations

within 20 min. Each end of the capillary, filled with

electrolyte, is immersed in a separate reservoir contain-

ing the same electrolyte and a high-voltage electrode.

0048CZE is strongly influenced by the degree of inert-

ness of the capillary, especially towards polar inter-

actions with buffer ions and/or analytes. Separations

by CZE in inert capillaries (e.g., Teflon) are based

only on the different mobilities of charged molecules

in an electric field, as in conventional zone electro-

phoresis. In contrast, CZE in noninert capillaries

(e.g., fused silica without a protective coating) uses

the separating forces of both electrophoretic migra-

tion and electroendosmotic flow.

0049In these cases, cations in the electrolyte (predomin-

antly protons) associate loosely with the capillary

wall, blocking the negative charges of the silanol

groups of uncoated fused silica. The hydrated cations

are pulled towards the cathode, inducing a bulk fluid

flow (the electroendosmotic flow, EEF) in that direc-

tion. The EEF can be strong enough to move all species

(positive, negative and neutral) in the same direction,

2060 ELECTROPHORESIS

though with different velocities. The EEF may ruin a

separation (e.g., by adsorption of the analyte to the

wall), but it can also be a crucial feature of the method.

0050 State-of-the-art fully automated CZE instruments

introduce samples automatically into the capillaries

either by electromigration or by hydrostatic or pneu-

matic methods. After zone electrophoresis in capil-

laries, separated sample components are detected in

real time as they pass an on-line detector. Ultraviolet

absorption and fluorescence detection have proven

most useful so far for CZE.

0051 CZE is faster and easier than slab gel electrophor-

esis. Although CZE is still in its infancy, powerful

separation methods have been already developed for

amino acids, peptides, nucleic acids, and their frag-

ments. In view of the increasing importance of bio-

technology in food manufacturing, CZE is expected

to play a major role in analytical food chemistry. (See

Amino Acids: Determination; Peptides.)

Application to Food Analysis/

Authentication

0052 Proteins and peptides are the primary targets of elec-

trophoresis in food analysis. Electrophoretic tech-

niques serve to characterize molecular architecture,

assess homogeneity, and identify and quantify pro-

teins.

0053 Electrophoresis has allowed food chemists to char-

acterize increases in protein yield, protein–protein

interactions, and enzymatic protein degradation. Fur-

ther studies of compositional parameters and process-

ing treatments have highlighted the importance of

these features on the flavor and texture of protein-

based foods. This is particularly true in the field of

dairy science and technology where the electrophor-

etic separation and quantification of milk proteins

and peptides have always helped new developments.

Figure 1 illustrates the simultaneous determination of

the major milk proteins by IEF. This efficient proced-

ure can be successfully applied to follow changes in

protein distribution in cheeses manufactured by ac-

celerated ripening and/or ultrafiltration procedures.

0054For studying proteins and larger peptides in com-

plex food systems, the most popular electrophoretic

techniques are isoelectric focusing and SDS (disc)

electrophoresis in ultrathin, horizontal PAG slabs.

Subtle differences in molecular properties such as

those due to posttranslational modifications or gen-

etic variation within protein classes can be deter-

mined with these methods. Perhaps the ultimate

in protein separation is obtained when a two-

dimensional electrophoretic approach is used. Truly

spectacular resolution can be achieved when one-

dimensional isoelectric focusing is combined with

two-dimensional SDS gradient PAGE, immuno-

diffusion, or immunoelectrophoresis. These high-

resolution techniques are used to follow proteolytic

breakdown and to verify the intact primary structure

of food proteins after certain processing treatments.

1 23456 789

fig0001 Figure 1 Simultaneous determination of the major milk proteins by isoelectric focusing in ultrathin-layer PAG. C1, a

-casein B;

C2, b-casein A

; C3, b-casein A

; C4, a

-casein; W1, a-lactalbumine; W2, b-lactoglobuline A; W3, b-lactoglobuline B. Samples from left:

recombined milk powders with casein: whey protein ratio of 100:0 (1), 98:2 (2), 95:5 (3), 90:10 (4), 80:20 (5), 70:30 (6), 60:40 (7), 50:50 (8),

and 0:100 (9). Reproduced from Electrophoresis, Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK

and Sadler MJ (eds), 1993, Academic Press.

ELECTROPHORESIS 2061

0055 It is not surprising that the discriminating power

and versatility of modern electrophoretic techniques

are unsurpassed in the positive identification of spe-

cific proteins and peptides. Thus, techniques such as

IEF, with or without immunodetection, provide reli-

able methods in food authentication in order to pre-

vent fraud and adulteration. Examples of current

interest include the verification/identification of the

species origin of meat, fish, milk and cheese products,

the detection of nonmeat and nonmilk proteins in

meat and milk products, respectively, the identifica-

tion of cereal varieties, the classification of pasta into

durum wheat and soft wheat types, the characteriza-

tion of cultivars of crops, fruits and vegetables, and

the determination of enzymes in beer and cheese. (See

Adulteration of Foods: Detection.)

0056 Isotachophoresis is the method of choice for the

analysis of complex mixtures of organic acids in

foods without derivatization but is also applicable

to artificial sweeteners and preservatives.

See also: Adulteration of Foods: Detection; Amino

Acids: Determination; Chromatography: High-

performance Liquid Chromatography; Enzymes: Uses in

Analysis; Immunoassays: Principles; Peptides; Protein:

Determination and Characterization

Further Reading

Andrews AT (1986) Electrophoresis – Theory, Techniques,

and Biochemical and Clinical Applications, 2nd edn.

Oxford: Clarendon Press.

Chrambach A (1985) The Practice of Quantitative Gel

Electrophoresis. New York: VCH.

Jorgenson JW (1986) Electrophoresis. Analytical Chemistry

85: 743A–760A.

Jorgenson JW and Philips M (eds) (1987) New Directions in

Electrophoretic Methods. Washington, DC: American

Chemical Society.

Westermeier R (1990) Elektrophorese-Praktikum. Wein-

heim: VCH.

Elimination Diets See Food Intolerance: Types; Food Allergies; Milk Allergy; Lactose Intolerance;

Elimination Diets

EMERGING FOODBORNE ENTERIC

PATHOGENS

D W K Acheson, University of Maryland School of

Medicine, Baltimore, MD, USA

Background

0001 Over the last 25 years, there have been an ever-growing

number of enteric pathogens linked with foodborne

disease (Table 1). Following some major food-

borne disease outbreaks in the early 1990s, there

has been increased focus on understanding the

causes and pathogenesis of foodborne disease. As

part of this initiative, federal agencies developed

improved surveillance systems and invested in

gaining a better understanding of how to prevent

foodborne disease through safer food processing.

During the course of this chapter, the initial section

will describe the current major emerging foodborne

infections in the context of our new epidemiological

knowledge. This is then followed by a more detailed

description of some of the principal emerging food-

borne infections that are considered to be important.

Clearly, different parts of the world have to deal

with different enteric pathogens, but the focus of

this chapter will be on issues within the USA and

other developed countries. It is not the goal of this

text to specifically address emerging foodborne

pathogens in developing countries that not only

have to deal with all the problems seen in

developed nations, but also frequently have to deal

with other specific pathogens.

Current Trends of Emerging Foodborne

Pathogens

0002In 1996, the Foodborne Diseases Active Surveil-

lance Network (FoodNet) was established. This is a

2062 EMERGING FOODBORNE ENTERIC PATHOGENS

cooperative venture between the Centers for Disease

Control, state health departments, the Food Safety

and Inspection Service of the US Department of

Agriculture, and the Food and Drug Administration.

FoodNet is a sentinel network that is designed to

produce national estimates of the burden and sources

of specific foodborne illnesses. FoodNet currently

focuses on obtaining information on nine important

and emerging foodborne infection agents (Table 2).

FoodNet operates by conducting population-based

active surveillance for confirmed cases of the various

pathogens shown in Table 2. Since the initiation of

FoodNet in 1996, the population under surveillance

has grown from five sites and some 14 million persons

in 1996 to 29.5 million that were under surveillance

during the year 2000. This number now represents

over 10% of the US population and involves a

number of states including Minnesota, Oregon,

California, Connecticut, Georgia, Maryland, New

York, and Tennessee.

0003 During the year 2000, there were 12 631 labora-

tory-confirmed cases of the nine diseases listed in

Table 2 at the FoodNet sites. Of these 12 631, 4640

were campylobacteriosis, 4237 were salmonellosis,

2324 were shigellosis, 631 were E. coli O157:H7,

484 were cryptosporidiosis, 131 yersiniosis, 101 lis-

teriosis, 61 Vibrios and 22 cyclosporiasis. Since 1996,

the levels of the major pathogens have varied amongst

the five original sites, and these data are shown in

Table 3. As can be seen from Table 3, there have been

minor fluctuations but little major change in the nine

important pathogens, although for Campylobacter

and Salmonella, the general trend is downward.

0004 One of the key criteria in relation to determining

the importance of emerging (and emerged) foodborne

pathogens relates to the severity and frequency of

disease with which they are associated. In the past,

there was a great deal of uncertainty about the

morbidity and mortality associated with foodborne

disease, but in 1999, a study was published by

the Centers for Disease Control that specifically

addressed these issues using FoodNet and other

data. The overall conclusion from this study was

that there was an estimated 76 million foodborne

illnesses per year, resulting in 325 000 hospitaliza-

tions and 5000 deaths. An interesting outcome of

this work was that known pathogens only accounted

for 14 million (18%) of these cases. This obviously

begs the question of what is going on in the remaining

80% of cases. Part of the answer lies in our inability

to diagnose foodborne disease in all cases. This is due

to a mixture of the failure of infected patients to seek

medical attention, the failure of physicians to order

stool cultures, and the failure of the laboratories to

find foodborne pathogens, even when they are pre-

sent, the latter either due to funding or technological

limitations. However, it does also raise the issue of

new emerging infectious foodborne agents that either

we are unaware of or have not yet been linked with

foodborne disease.

0005Whereas there are over 200 known diseases trans-

mitted via food, including microbes, toxins, chemicals,

physical hazards, and prions, there is a relatively short

list of the important agents, and many of these could

be considered as emerging simply because they were

not recognized until recently. Mead et al. determined

tbl0001 Table 1 Emerging foodborne pathogens since 1977

Campylobacter jejuni

Cryptosporidium parvum

Cyclospora cayentanesis

Shiga toxin-producing E. coli

Listeria monocytogenes

Norwalk-like viruses

Salmonella enteritidis

Salmonella typhimurium DT104

Transmissible spongiform encephalopathy

Vibrio cholerae O139

Vibrio vulnificus

Vibrio parahemolyticus

Yersinia enterocolitica

tbl0002Table 2 Foodborne pathogens routinely under surveillance as

part of FoodNet

Bacteria

Campylobacter spp.

Escherichia coli O157:H7

Listeria monocytogenes

Salmonella spp.

Shigella spp.

Vibrios

Ye r sini a

Protozoa

Cryptosporidium

Cyclospora

tbl0003Table 3 Incidence (per 100 000 population) of diagnosed

infections in the original five FoodNet sites

Pathogen 1996 1997 1998 1999 2000

Campylobacter 23.5 25.2 21.4 17.5 20.1

Cryptosporidium nr 3.7 2.9 1.8 2.4

Cyclospora nr 0.4 0.1 0.1 0.1

Escherichia coli O157 2.7 2.3 2.8 2.1 2.9

Listeria 0.5 0.5 0.6 0.5 0.4

Salmonella 14.5 13.6 12.3 13.6 12.0

Shigella 8.9 7.5 8.5 5.0 11.6

Vibrio 0.2 0.3 0.3 0.2 0.3

Ye r sini a 1.0 0.9 1.0 0.8 0.5

nr, not reported.

Modified from Preliminary FoodNet Data on the Incidence of Foodborne

Illness – Selected Sites, United States, 2000 (2001) MMWR 50: 241–246.

EMERGING FOODBORNE ENTERIC PATHOGENS 2063

which are the important foodborne pathogens in

terms of frequency of disease, and at the same time

determined what proportion of illness due to those

pathogens were actually transmitted via food, as

opposed to some other means of transmission such

as person-to-person or directly from animals. A sum-

mary of this information is presented in Table 4 and

illustrates the point that some of the most common

foodborne infections such as Campylobacter and

Norwalk-like virus result in a relatively small number

of hospitalizations and death compared with other

pathogens such as Listeria monocytogenes. Another

key observation was that five pathogens accounted

for more than 90% of the deaths as follows: Salmon-

ella – 31%; Listeria – 28%; Toxoplasma – 21%;

Norwalk-like virus – 7%; Campylobacter – 5%;

E. coli O157:H7 – 3%.

0006 Generally, there is a perception amongst many that

foodborne disease usually occurs as part of outbreaks

(defined as an incident in which two or more persons

experience an illness resulting from the ingestion of a

common food). In fact, the vast majority of food-

borne disease are isolated cases that are not linked

to other cases. A recently published summary of

documented outbreaks, which occurred between

1993 and 1997, illustrates the important emerging

pathogens and foodborne agents that are most fre-

quently linked to outbreaks. During that period, there

were 2751 outbreaks investigated by the Centers for

Disease Control averaging about 500 outbreaks per

year. A total of 86 058 people were sick in connection

with the outbreaks, and while only 32% had a known

etiology, they accounted for 59% of the 86 058 infec-

tions. Table 5 summarizes some of the outbreak data

and illustrates the importance of Salmonella and

E. coli as causes of outbreaks and death, but also

demonstrates the importance of two preformed

toxins, Scrombotoxin and Ciguatoxin, as causes of

outbreaks. As already stated, outbreaks account for

the minority of foodborne disease. Based on previous

work and assuming 76 million cases of foodborne

disease per year, during the 5 years from 1993 to

1997, there would have been 380 million episodes

of foodborne disease. During this time, only 86 058

cases were linked with outbreaks.

0007In summary, since 1996 and the inception of Food-

Net, we have learnt a great deal about the foodborne

agents that are responsible for disease and death in

the USA. Many of these were unrecognized 20 or 30

years ago and underscore the need to remain vigilant

about new agents and newly emerging pathogens.

Specific Emerging Foodborne Pathogens

0008In the following sections, a more detailed description

of some of the specific emerging foodborne pathogens

is given. It is beyond the scope of this text to discuss in

detail all the emerging foodborne enteric pathogens,

but readers are referred to other specific chapters in

this text that deal in more detail with individual

microbes.

Campylobacter

0009Foodborne disease due to Campylobacter, which was

not recognized until the mid-1970s, is now one of the

most frequently diagnosed causes of food-related

gastroenteritis. In fact, Campylobacter and Salmon-

ella probably account for 70% of diagnosed cases

of bacterial foodborne disease in the USA.

tbl0004 Table 4 Ranking of the most common microbial causes of foodborne disease with an estimate of the percentage of infections that

are transmitted via food, and deaths as a percentage of total foodborne disease-related deaths

Disease oragent Estimated totalcases Percentagefoodborne transmission Percentage of deaths

Norwalk-like viruses 23 000 000 40 7

Campylobacter spp. 2 500 000 80 5

Giardia lamblia 2 000 000 10 0.1

Salmonella 1 400 000 95 31

Shigella 450 000 20 0.8

Cryptosporidium parvum 300 000 10 0.4

Clostridium spp. 250 000 100 0.4

Toxo p l a sma gon d ii 225 000 50 21

Staphylococcal food poisoning 185 000 100 0.1

Shiga toxin-producing E. coli 110 000 85 1.4

Yersinia enterocolitica 100 000 90 0.1

Bacillus cereus 27 000 100 0

Cyclospora cayetanensis 16 000 90 0

Listeria monocytogenes 2 500 99 28

Expressed as a percentage of the number of deaths related to foodborne pathogens.

Modified from Mead PS, Slutskev L, Dietz V et al. (1999) Food-related illness and death in the United States. Emerging Infectious Diseases 5: 607–625.

2064 EMERGING FOODBORNE ENTERIC PATHOGENS

0010 There are two main species of Campylobacter that

are responsible for most of the illness seen in humans.

C. jejuni accounts for the vast majority (* 90%), and

C. coli accounts for the majority of the remainder.

Other Campylobacter species that have been associ-

ated with gastroenteritis in humans include C. fetus,

C. upsaliensis, C. hyointestinalis,andC. lari. Cam-

pylobacter species are fragile organisms and tend to

die in transport media, so rapid plating of samples is

an advantage. Enriched media are usually used that

contain a variety of antibiotics to reduce competitive

flora. The infectious dose of Campylobacter seems to

be variable depending on the strain and the host, but

low numbers of Campylobacter may be all that is

needed to cause disease, and in some instances, in

human volunteers, less than 100 organisms can

cause disease. These organisms are more frequently

associated with sporadic disease rather than out-

breaks, and person-to-person spread does not appear

to be common, although it can occur in a family

environment, for example. This sporadic epidemi-

ology suggests that the infectious dose may actually

be higher in many situations. C. jejuni and C. coli are

commensals in the intestines of many animals and

birds, including domestic pets. The main vehicles for

infection are raw meats, especially poultry, milk, and

water. Surface water is frequently contaminated

with campylobacters, and waterborne outbreaks

have occurred.

0011 Campylobacter species are invasive organisms,

which means that they have the capacity to invade

the epithelial cells lining the intestine. The pathogen-

esis of Campylobacter is dependent on its motility.

In vitro, nonmotile strains are not capable of invading

intestinal epithelial cells. It is assumed that invasion

in the intestine is one of the principal pathogenic

mechanisms of these bacteria, although the genes

involved are not known. The recent publication of

the C. jejuni genome is, however, helping with the

discovery of new genes and determining the function

of previously identified genes.

0012Following infection with campylobacters, there

is typically an inflammatory response with marked

inflammatory infiltration of the lamina propria

resulting in leukocytes being present in the stools.

Clinically, symptoms usually occur within 2–3 days

following exposure, or up to 7 days after exposure.

Nongastrointestinal symptoms such as fever (which

may be high), headache, and myalgia may precede the

onset of nausea, vomiting, and diarrhea. Diarrhea is

usually the predominant symptom. This may be

watery or bloody, and is usually associated with

severe cramping abdominal pain. Interestingly, the

disease is occasionally biphasic with an apparent set-

tling of symptoms after 4–5 days, only to be followed

by a recrudescence. A number of complications are

associated with Campylobacter, including cholecysti-

tis, hepatitis, acute appendicitis, pancreatitis, and

focal extraintestinal infections. Longer-term compli-

cations include reactive arthritis, Reiter’s syndrome,

uveitis and Guillain–Barre

syndrome; molecular

mimicry may be involved in some of these compli-

cations. The majority of Campylobacter infections

are self-limiting and require supportive therapy only,

especially in the form of rehydration. Antibiotic ther-

apy is not routinely required, but patients with pro-

longed or severe symptoms or who have other

significant risk factors (AIDS, cirrhosis, diabetes,

etc.) should be treated. Erythromycin is a reliable

therapy, although there is a general move toward

using quinolone antibiotics. Despite the use of ery-

thromycin for many years, the resistance levels have

remained low. This is not the case with the quinolones,

and there are increasing reports of ciprofloxacin

tbl0005 Table 5 Major causes of foodborne disease outbreaks in the USA between 1993 and 1997, showing the numbers of outbreaks, the

numbers of total cases, and the number of deaths associated with a particular etiology

Etiology Number of outbreaks (%) Number of cases(%) Number of deaths (%)

Salmonella 357 (13.0) 32 610 (37.6) 13 (44.8)

Escherichia coli 84 (3.1) 3 260 (3.8) 8 (27.6)

Scrombotoxin 69 (2.5) 297 (0.3) 0

Ciguatoxin 60 (2.2) 205 (0.2) 0

Clostriduim perfringens 57 (2.1) 2 772 (3.2) 0

Shigella 43 (1.6) 1 555 (1.8) 0

Staphylococcus aureus 42 (1.5) 1 413 (1.6) 1 (3.4)

Campylobacter 25 (0.9) 539 (0.6) 1 (3.4)

Hepatitis A 23 (0.8) 729 (0.8) 0

Bacillus cereus 14 (0.5) 691 (0.8) 0

Clostridium botulinum 13 (0.5) 56 (0.1) 1 (3.4)

Norwalk virus 9 (0.3) 1 233 (1.4) 0

Listeria monocytogenes 3 (0.1) 100 (0.1) 2 (6.9)

Modified from Centers for Disease Control (2000) Surveillance for foodborne disease outbreaks – United States, 1993–1997. MMWR 49: 1–51.

EMERGING FOODBORNE ENTERIC PATHOGENS 2065

resistance, which may become more of a problem in

view of the expanded use of these antibiotics in agri-

culture.

Listeria monocytogenes

0013 L. monocytogenes is a Gram-positive motile rod that

is one of the most frightening foodborne pathogens

because of the high mortality rate associated with

infection. Of the seven Listeria species, only L. mono-

cytogenes is pathogenic for humans. L. monocyto-

genes is a common environmental organism and is

frequently present in soil and water, on plants, and

in the intestinal tracts of many animals. It has been

found in 37 different types of mammals and at least

17 species of birds. Between 1 and 10% of people

are carriers of L. monocytogenes. This organism is

associated with both sporadic disease and outbreaks.

Incriminated foods include milk, cheese, raw vege-

tables, undercooked meat, and foods prepared for

instant use such as hot dogs. The infectious dose is

not really known: some studies have suggested that it

may be very high (up to 10

organisms), and others

have suggested that it may be as low as several hun-

dred. In practice, the most critical aspect is probably

individual susceptibility rather than the infectious

dose per se. Clinically, it usually begins with non-

specific symptoms such as fever, myalgia and gastro-

intestinal upset in the form of diarrhea, and

nausea. What makes L. monocytogenes exceptional

as a foodborne pathogen is its very high mortality

rate. Of the approximately 1800 cases per year that

are estimated to occur in the USA, there are over 400

deaths. This gives a case fatality rate of over 20%.

There are certain groups that are especially at risk of

developing listeriosis, and these include pregnant

women, the elderly, and the immunocompromised.

Transplacental transmission in pregnant women is a

major concern, although it does not inevitably lead to

major consequences. Spontaneous abortion, prema-

turity, neonatal sepsis, and meningitis are all compli-

cations of transplacental transmission. Although

L. monocytogenes is readily killed by heat and

cooking, the fact that it is so ubiquitous makes recon-

tamination a real risk. This then poses a major health

problem, because the organism will grow and multi-

ply at standard refrigerator temperatures. Thus, even

minor contamination of a product may, after storage,

result in high levels of bacteria, even if the product

has been adequately refrigerated. Following diagno-

sis, L. monocytogenes is readily treated by penicillins

or aminoglycosides. L. monocytogenes has also been

linked with acute gastroenteritis in individuals

with normal immune systems, but it is unclear how

frequently this occurs.

Salmonella

spp.

0014Salmonella are one of the most common causes of

foodborne illness in humans, and although they are

not as a group considered to be emerging foodborne

pathogens, some types of Salmonella (e.g., S. enteri-

tidis in eggs) have definitely emerged as a major prob-

lem in recent years. There are many types of

Salmonella, but they can be divided into two broad

categories: those that cause typhoid and those that do

not. The typhoidal Salmonella such as S. typhi and

S. paratyphi only colonize humans and are usually

acquired by the consumption of food or water con-

taminated with human fecal material. The much

broader group of nontyphoidal Salmonella are

found in the intestines of other mammals and there-

fore are acquired from the consumption of food or

water that has been contaminated with fecal material

from a wide variety of animals and poultry.

0015The number of cases of nontyphoidal Salmonella

has increased steadily over the last four decades.

S. entertidis particularly has become a growing prob-

lem, especially in hen eggs. In the year 2000 FoodNet

data, of the 4237 Salmonella that were isolated, 3686

were serotyped, and 23% were S. typhimurium, and

15% were S. enteritidis. Currently, the estimate is that

around 1 in 10 000 eggs are contaminated with

Salmonella. It is now known that Salmonella can

penetrate intact eggs lying in fecally contaminated

material and also infect eggs transovarially during

egg development before the shell is formed. Other

than eggs, common sources of nontyphoidal salmon-

ellosis are milk, foods containing raw eggs, meat and

poultry, and fresh produce. Essentially, as with many

of the other foodborne bacterial infections, Salmon-

ella are frequently transmitted through fecal contam-

ination of food because of the large numbers of

animals that carry the organism. The infective dose

of nontyphoidal Salmonella may vary from < 100 to

, depending on the host and on the actual type

of Salmonella. Irrespective of the type of Salmonella,

the most critical virulence determinant of these bac-

teria is their ability to invade the intestinal epithe-

lium, following which they interact with underlying

lymphoid tissue.

0016Clinically, nontyphoidal Salmonella typically pre-

sents with gastroenteritis 24–48 hours after exposure

to the organisms. There is usually nausea, vomiting,

abdominal cramps, and diarrhea, which may be

watery or occasionally bloody. It is not unusual for

there to be associated symptoms of fever, chills, head-

ache, and myalgia. Occasionally there can be long-

term consequences following Salmonella infection

such as reactive arthritis (especially in individuals who

are HLA B27-positive), endocarditis, and localized

2066 EMERGING FOODBORNE ENTERIC PATHOGENS