Vol.18, No.

1 January 1996 V

Continuing Education Article

Endotoxin and Disease

FOCAL POINT
in Food Animals*
★ Conditions ranging from diarrhea to University of California
life-threatening meningoencephalitis
James S. Cullor, DVM, PhD
are caused by gram-negative
bacteria in food animals; current W. L. Smith, BS, MT
treatments must be improved by
an enhanced understanding of the

G
molecules responsible for the ram-negative bacteria are responsible for many clinical conditions ob-
pathophysiologic changes. served in food animal species. Current treatments and management
practices are only moderately successful in ameliorating the clinical con-
■ sequences of gram-negative infections. Future research and clinical approaches to
KEY FACTS managing these devastating diseases depend on a better understanding of the ori-
gin of endotoxin and how various cells and organ systems react to its presence in
■ A detailed description of the
the body. This article considers clinically relevant information regarding the
endotoxin complex includes
sources, structure, and biologic activities of endotoxin.
portions of the cell membrane,
cell envelope, lipoprotein, outer
COMPONENTS OF THE GRAM-NEGATIVE
membrane, lipopolysaccharide,
BACTERIAL ENDOTOXIN COMPLEX
bacteria capsule, and glycocalyx.
Endotoxins have been demonstrated to be localized on the surface of bacteri-
al cells and to form (together with phospholipids and proteins) the outer mem-
■ Diseases in food animals that are
brane of gram-negative bacteria (Figure 1). A detailed description of the endo-
associated with gram-negative
toxin complex should include various portions of the cell membrane, the cell
organisms include neonatal
envelope, lipoprotein, outer membrane, lipopolysaccharide (LPS), bacteria cap-
coliform septicemia, coliform
sule, and glycocalyx.1
mastitis, salmonellosis, and
The cell envelope is composed of the macromolecular layers that surround
pneumonia.
the bacterium. In gram-negative bacteria, the envelope includes a cell mem-
brane, a cell wall, and possibly a capsule and/or a glycocalyx layer.
■ Morbidity and mortality
Gram-negative bacteria possess a typical cell membrane that is composed of
associated with gram-negative
phospholipids and proteins. The structure contains the cytochromes and en-
bacterial sepsis, in part, are
zymes involved in electron transport and oxidative phosphorylation. This por-
a result of host reactions to
tion of the bacteria also contains chemoreceptors and is the site of action of
bacterial cell wall components.
certain antibiotics (e.g., polymyxin).
The cell wall is the portion of the cell envelope that is external to the cyto-
■ Veterinarians should routinely
plasmic membrane and internal to the capsule or glycocalyx. In gram-
reevaluate immunization
negative bacteria, the cell wall is composed of peptidoglycan, lipoprotein, the
protocols to minimize the
outer phospholipid membrane, and lipopolysaccharide. This structure gives the
exposure of food animals to
organism osmotic protection and gram-staining characteristics.
endotoxin. *Supported in part by USDA formula funds, the Livestock Disease Research Laborato-
ry, and the California Milk Advisory Board. All funds administered by the School of
Veterinary Medicine, University of California, Davis, California.
Food Animal
Peptidoglycan (also re-
ferred to as mucopeptide or
murein) is found in all bac-
terial cell walls. It is a com-
plex polymer that consists
of a backbone and a set of
identical tetrapeptide side
chains. Peptidoglycan is the
site of action of certain an-
tibiotics, including peni-
cillin and cephalosporins.
Lipoprotein provides a
strong cross-link between
the peptidoglycan and out-
er membrane. The outer
membrane is a phospho-
lipid bilayer in which the Figure 1—Schematic representation of the components of presence of partial struc-
phospholipids of the outer gram-negative bacteria (LPS = lipopolysaccharide).
portion are replaced by
lipopolysaccharides. This
structure is responsible for protecting the cells from
harmful enzymes and preventing leakage of periplasmic
proteins.
Lipopolysaccharide is found in gram-negative bacte-
ria and consists of lipid-A (several long-chain fatty acids class of bacteria by forming the endotoxin complex that
attached to phosphorylated glucosamine disaccharide
units), a polysaccharide composed of a core and termi-
nal repeating units, and major surface antigens (includ-
ing the O antigen, found in the polysaccharide compo-
nent). The lipopolysaccharide is also referred to as
endotoxin, and the lipid-A portion is prominently asso-
ciated with bacterial toxicity.
The capsule is a well-defined structure of polysaccha-
ride that surrounds the bacterial cell and is generally lo-
cated external to the cell wall. The structure contributes
to bacterial invasiveness because it provides bacterial re- septicemia, coliform mastitis, salmonellosis, pneumo-
sistance to phagocytosis.
The term glycocalyx refers to a loose network of
polysaccharide fibrils that surrounds some bacterial cell
walls; the glycocalyx is sometimes referred to as the
slime layer of the bacteria. It is associated with many
adhesive properties of the bacterial cell and contains
prominent antigenic sites.
Two other commonly mentioned portions of gram-
negative bacteria that are not considered a part of the
endotoxin complex are the flagella and pili (fimbriae).
Flagella are protein appendages used by the bacteria for
locomotion. They may cover the entire bacterial cell
surface or be located only in one area of the cell. The
flagella are composed of a protein subunit (called flag-
ellin) that may provide many antigenic determinants.
The pili are rigid surface appendages primarily made of
protein molecules with antigenic properties. The ordi-
LIPID-A ■ MAJOR SURFACE ANTIGENS ■ TUMOR NECROSIS FACTOR ■ SEPTICEMIA
The Compendium January 1996
nary pili are involved in
bacterial adherence.
LIPID-A
Lipopolysaccharides con-
stitute the O antigens and
endotoxins of gram-nega-
tive bacteria. The structure
of lipid-A from various bac-
teria has been described
and chemically synthe-
sized.2–4 Through this pro-
cess, some of the intrinsic
heterogeneity of the lipid-A
molecule has been deter-
mined to be related to the
tures resulting from incom-
plete synthesis. Lipid-A
constitutes the covalently
linked lipid component of lipopolysaccharide and is the
endotoxic moiety of gram-negative bacteria.
The polysaccharide and lipid portions of lipopoly-
saccharide contribute to the pathogenic potential of this
provides the mediator shock potential for the disease pro-
cess. The interaction of the lipid-A molecule with various
cell types may elicit the secretion of bioactive media-
tors—such as tumor necrosis factor (TNF), interleukin-1
(IL-1), and interleukin-6 (IL-6)—which can lead to sys-
temic mediator shock events.
GRAM-NEGATIVE BACTERIAL DISEASE
Gram-negative organisms are responsible for many
diseases in food animals, including neonatal coliform
nias caused by Pasteurella and Actinobacillus species,
brucellosis, metritis, campylobacteriosis, infections of
the cornea and sclera, and thromboembolic meningoen-
cephalitis. Epidemiologic studies in food animal neo-
nates (e.g., cattle, pigs, and small ruminants) demon-
strate that most clinical infections and mortality in this
age group result from gram-negative organisms, specifi-
cally Escherichia coli and Salmonella species.5–10 Mortali-
ty varies among farms and production units; estimates
of dairy calf death losses during the first eight weeks of
life range from 2.5% to 29%.11
Septicemia caused by E. coli (colisepticemia) generally
affects calves younger than 1 week of age and is charac-
terized by a rapidly fatal disease course. Low serum im-
munoglobulin concentration and exposure to invasive
serotypes of E. coli are major determinants of the devel-
opment of colisepticemia.7,12–15 Death losses from salmo-
The Compendium January 1996
nellosis in cattle are most severe in confinement-raised
dairy calves at 1 to 10 weeks of age.9,10 Although salmo-
nellosis is usually described as being confined to the gas-
trointestinal tract, most calves develop bacteremia with
spread of the infection to liver, lungs, bone marrow, and
central nervous system.
In older cattle, shipping fever pneumonia (SFP) oc-
curs in feedlot animals; 75% of cases develop during
the first 45 days that the cattle are housed at the feedlot
facility. The disease process is apparently caused by a
complex interaction among stressors, viruses, and bac-
teria that have an endotoxin component in the cell
wall. The primary bacterial isolates of shipping fever
pneumonia are Pasteurella hemolytica, P. multocida,
and some Pseudomonas species.16 The capsules of P.
hemolytica, P. multocida, and Haemophilus somnus
contain lipopolysaccharide (endotoxin). The release of
the endotoxin molecule induces several events, includ-
ing initiation of complement and coagulation cascades
and recruitment of activated neutrophils and macro-
phages. Pasteurella hemolytica produces a protein cyto-
toxin that is lethal to these macrophages and neu-
trophils; enzymes that can destroy tissue are thus
released into the microenvironment. In addition, reac-
tive oxygen intermediates are produced that are capable
of destroying neutrophils and surrounding tissue.
Coliform mastitis, another disease process initiated
by gram-negative opportunists, can be devastating to
dairy production units. The process, which primarily
develops during the first 100 days of lactation but may
occur at any stage of lactation or during the dry period,
encompasses all degrees of severity from peracute to
subclinical. 17 The bacteria most frequently isolated
from this form of bovine mastitis include E. coli, En-
terobacter aerogenes, and Klebsiella pneumoniae. Other
gram-negative organisms that are less commonly isolat-
ed include Pseudomonas aeruginosa, P. multocida, and
Serratia marcescens. The resultant bacterial growth in
the mammary gland can cause serious local and sys-
temic consequences.18–21
CLINICAL SIGNS ASSOCIATED
WITH DISEASE: MEDIATOR SHOCK
Morbidity and mortality associated with gram-nega-
tive bacterial sepsis are apparently the consequences of
host reaction to bacterial cell wall components (e.g., en-
dotoxin, the lipopolysaccharide cell wall component of
gram-negative bacteria).22–24 The following endogenous
and exogenous factors, however, have been linked to the
pathophysiology of sepsis and mediator shock: (1) endo-
toxin from gram-negative bacteria; (2) peptidoglycan
and exotoxins from gram-negative bacteria; (3) endotox-
in-binding proteins and receptors; (4) bactericidal pro-
SHIPPING FEVER PNEUMONIA ■ COLIFORM MASTITIS ■ CYCLOOXYGENASE PATHWAY
Food Animal
teases; (5) release of cytokines, histamine, bradykinin,
and arachidonic acid metabolites; (6) complement acti-
vation; and (7) endothelium-derived adhesion molecules.
The arachidonic acid metabolites originate in the cy-
clooxygenase and lipoxygenase pathways. Members of
the cyclooxygenase pathway and their biologic activities
include PGE2 (vasodilator), PGF2 (vasoconstrictor),
thromboxane A 2 (vasoconstrictor and promoter
of platelet aggregation), and PGI 2 (vasodilator and
inhibitor of platelet aggregation). Members of the lipoxy-
genase pathway (leukotrienes) include 5-hydroxyeico-
satetraenoic acid (5-HETE) and leukotrienes (LT) A4,
B4, C4, and D4. These compounds are potent bron-
choconstrictors and vasoconstrictors, elicit plasma exuda-
tion, are chemotactic for leukocytes, and are involved in
microthrombus formation.
Variations in the biologic activity of endotoxins have
been observed. These differences relate to the presence
of lipid-A–associated protein, aggregation, polysaccha-
ride composition, culture conditions, and the source of
the organism. Many of the clinical signs observed in
conjunction with gram-negative bacterial disease have
been reproduced experimentally by administering puri-
fied endotoxin (also known as lipopolysaccharide) in
various doses and by various routes. The effects of
lipopolysaccharide on host cells (e.g., macrophages,
platelets, and endothelial cells) and on the release of in-
flammatory mediators also influence the clinical signs
observed at various stages of the disease process. For
example, the severity of inflammatory responses after
lipopolysaccharide challenge has been demonstrated to
vary directly with receptor numbers on the macrophage
(which can vary among animals).25
The general effects of endotoxins are well chronicled
and reportedly include lethargy, respiratory distress, tran-
sitory hyperthermia followed by hypothermia, decreased
systemic blood pressure, increased heart rate followed by
decreased cardiac output, diarrhea, changes in blood cell
counts, and alterations in the blood coagulation system.
Some of the more specific physiologic and pathologic re-
actions are lymphopenia followed by lymphocytosis, neu-
tropenia followed by leukocytosis, hyperglycemia fol-
lowed by hypoglycemia, depletion of liver glycogen,
anabolic and catabolic responses in protein metabolism,
localized and generalized Shwartzman reactions (i.e., local
thrombosis formation and/or general disseminated in-
travascular coagulation with bilateral renal cortical necro-
sis), induction of transitory tolerance to further endotoxin
insults, and altered reproductive performance (see the
box).
The classical and alternative complement pathways are
activated, resulting in generation of anaphylatoxin and
many secondary local and systemic effects. The intrinsic
Food Animal
and extrinsic pathways of the
Effects of clotting system also are acti-
Endotoxins vated, expression of tissue
factor is enhanced, and dis-
General seminated intravascular coag-
ulation may ensue. Platelets
■ Lethargy
aggregate and sequester in
■ Respiratory distress various capillary locations and
■ Transitory secrete their mediators. Neu-
hyperthermia followed trophils respond by produc-
by hypothermia ing inflammatory mediators
■ Decreased systemic (prostaglandins or leuko-
trienes) and oxygen radicals.
blood pressure
Macrophage function is en-
■ Increased heart rate hanced, and the cells secrete
followed by decreased cytokines. Comprehensive
cardiac output discussions of the pathophysi-
■ Diarrhea ologic effects of endotoxins in
■ Changes in blood cell ruminants are available in the
literature.26,27
counts
■ Alterations in the blood ANTIBIOTIC THERAPY
coagulation system AND ENDOTOXIN
RELEASE
Specific Antibiotic-induced release
■ Lymphopenia followed of endotoxin has been of
clinical and research interest
by lymphocytosis
for some time. A 3- to 78-
■ Neutropenia followed fold increase in the total con-
by leukocytosis centration of endotoxin in
■ Hyperglycemia vitro and in vivo has been re-
followed by ported. 28,29 There is appar-
hypoglycemia ently considerable overlap
between the effect of β-lac-
■ Depletion of liver
tam antibiotics and non–β-
glycogen lactam antibiotics, with an
■ Anabolic and catabolic unexplained delay between
responses in protein the lethal activity of antibi-
metabolism otics and the release of endo-
■ Schwartzman reactions toxin. The lytic and nonlytic
release of endotoxin thus
■ Transitory tolerance to
must be considered in the
further endotoxin pathogenesis of disease and
insults will influence the therapeutic
■ Altered reproductive efficacy of antiendotoxin
performance therapy. Scientific research is
necessary concerning this
topic in food animals.
ENDOTOXINS, EXOTOXINS,
AND ENTEROTOXINS
Endotoxins are heat-stable lipopolysaccharide–
lipoprotein complexes that may be released during cell
ANTIBIOTIC-INDUCED RELEASE ■ ANTIENDOTOXIN THERAPY ■ HEAT-LABILE PROTEINS
The Compendium January 1996
growth and bacterial lysis as part of the outer membrane
of gram-negative bacteria. The release of this compound
results in the initiation of mediator cascades (e.g., release
of cytokines, serotonin, histamine, bradykinin, and
arachidonic acid metabolites) that culminate in the clas-
sical clinical signs mentioned. Although it is a strong py-
rogen in the host, endotoxin is weakly toxic, rarely fatal
compared with exotoxins, and a relatively poor immuno-
gen. No toxoid preparation, in the classical scientific def-
inition, is provided in most vaccine preparations of this
molecule. The appropriate definition of a toxoid dictates
that the endotoxin molecule in the preparation be treat-
ed by chemicals or heat in such a way as to eliminate the
toxic qualities while retaining the antigenic properties.
These data are often unavailable to the public.
Exotoxins are heat-labile proteins excreted by certain
gram-positive or gram-negative bacteria. The molecules
possess a specific mode of action (e.g., cytotoxin, entero-
toxin, or neurotoxin) with defined actions on cells or tis-
sue. Exotoxins are highly toxic and often result in a fatal
disease process. Compared with endotoxins, these bacte-
rial proteins are highly immunogenic and stimulate the
production of neutralizing antibody (antitoxin). Treating
the protein toxin with formaldehyde eliminates its toxici-
ty without destroying the immunogenic properties.
Formaldehyde treatment of endotoxin does not make the
lipopolysaccharide molecule a toxoid as strictly defined.
Exotoxins reportedly do not produce fever in the host.
Enterotoxins are exotoxins that specifically affect the
small intestine, causing changes in intestinal permeabil-
ity that lead to diarrhea. The substantial diarrhea ob-
served in patients with cholera is due to the action of
this type of toxin and is commonly caused by food-
poisoning microorganisms.
ESCHERICHIA COLI 0157:H7 AND VEROTOXINS
During the winter of 1993, a severe outbreak of food-
borne human disease in the Pacific Northwest was
linked to microbial contamination of ground beef with
E. coli 0157:H7.30 The outbreak occurred in several lo-
cations, and the number of cases reached 400. Of these,
125 patients were hospitalized. At least 29 patients de-
veloped acute renal failure, and all but 8 of these re-
quired hemodialysis. Three young children died. Al-
though this outbreak received deserved public attention,
it is not unique. Escherichia coli 0157:H7 was first iden-
tified in 1982, when it was determined to be the cause
of a multistate outbreak of hemorrhagic colitis associat-
ed with hamburger patties sold by a national fast-food
chain.31 Other recent outbreaks of E. coli 0157:H7–
related disease have been associated with contaminated
apple cider, unpasteurized milk, mayonnaise, and mu-
nicipal water supplies.
The Compendium January 1996
Because some of the most significant clinical events
have involved contaminated meat, the public view of
food-borne disease and the safety standards required to
prevent its occurrence has changed. Veterinarians should
be active in improving on-farm, preharvest, food safety
quality assurance programs to prevent contamination of
milk and meat by pesticides, herbicides, hormones, an-
tibiotics, and microbes.
The vast majority of strains of E. coli isolated from
feces are part of the normal intestinal flora. They play
an important role in maintaining optimum intestinal
physiology. In this group of bacteria, however, are
strains that are pathogenic and cause diarrhea. Strains
that cause diarrhea do so by mechanisms that have re-
sulted in the following classifications: enteropathogenic
(EPEC), enterotoxigenic (ETEC), enteroinvasive
(EIEC), and verotoxigenic (VTEC).32 Canadian inves-
tigators have demonstrated that toxins produced by
strains of E. coli serotype 0157:H7 are cytotoxic for
vero cells; hence the term verotoxins. In addition, isola-
tion of the pathogen was closely related with hemor-
rhagic colitis (HC) syndrome in humans.
Two clinically important verotoxins produced by E.
coli (VT1 and VT2) are members of a family of many
similar cytotoxins. The verotoxins are subunit toxins con-
stituted by an A (active) subunit and several B (binding)
subunits. The verotoxins bind to the receptor on the sur-
face of an intestinal cell via the B subunit. The A subunit
is then taken into the cell and cleaved to an active frag-
ment that inhibits cellular protein synthesis. Escherichia
coli 0157:H7 is often the most frequent serotype of
VTEC isolated. The reported predominance of serotype
0157 is undoubtedly biased by the wide use of methods
adapted only for this serotype. More than 57 other
serotypes that produce verotoxins have been described.33
A German study used a common biotechnology
technique (DNA–DNA colony hybridization using
specific gene probes for VT1 and VT2) to examine
2100 E. coli strains from the feces of healthy animals.
Ten of 82 milk cows, 20 of 212 beef cattle, and 5 of 75
pigs reportedly carried genes for VT1 and/or VT2.
Several of the serotyped isolates have been described to
be pathogenic in humans (0157:H7, 082:H8, 0116,
0113, 0126, and 091).34
In a portion of the National Dairy Heifer Evaluation
Project conducted by Veterinary Services (USDA/
APHIS), 6894 heifer calves in 1068 dairy herds were
sampled in 28 states. The study reported a prevalence of
isolation of E. coli 0157:H7 in calves of 3.6 per 1000.
Escherichia coli 0157:H7 was found among calves from
2 weeks to greater than 12 weeks of age; however, no cul-
ture-positive feces were found among 633 calves sampled
during the first week of life. Culture-positive calves were
CONTAMINATED MEAT ■ VEROTOXINS ■ CULTURE-POSITIVE FECES ■ IMMUNOGENS
Food Animal
present in all regions of the United States; the herd
prevalence was estimated at 5%.35 No information was
reported concerning the capability of these isolates to
produce verotoxins.
VACCINES AND ENDOTOXIN CONTENT
Traditional gram-negative vaccine preparations have
been plagued by problems of adverse reactions in the
host species, thus earning the distrust of many veterinari-
ans and producers. The Limulus amebocyte lysate (LAL)
test was used to determine endotoxin levels (endotoxin
units [EU/ml]) present in commercial vaccine prepara-
tions. The usual conversion of 5 EU/ng of endotoxin ap-
plies to all of the figures in this report. Commercial
gram-negative immunogens contain thousands of
EU/ml of vaccine and may contain millions of EU/ml of
free endotoxin, as measured by the LAL (Table I).
Because the pyrogenic threshold for pharmaceutical
compounds is 5 EU/kg body weight, the maximum tar-
get amount in a 700-kg cow would be 3500 EU. No py-
rogenic thresholds have been established for food animals
related to vaccine administration. Many of the immu-
nization schedules used today in food animals may exceed
the target amount set in the pharmaceutical compound
example. As producers become more aware of the adverse
reactions that may result from immunization protocols,
the veterinarian in charge of herd health programs must
be aware of the endotoxin levels in the vaccines being ad-
ministered. For example, the veterinarian may need to
have the products tested and then weigh safety and effica-
cy considerations in selecting the immunogen to be ad-
ministered or the frequency of vaccine administration. To
date, no published research studies have assessed either of
these strategies using commercial vaccine preparations.
EXPERIMENTAL FINDINGS
An experiment of importance to the dairy industry
involved the administration of commercially available
immunogens to lactating dairy cattle and the effect of
this practice on milk production during the next few
days (Table II). The immunogens were administered
via the dose and route recommended by the manufac-
turer and were administered alone or in combination
with the other two vaccines. The subjects were lactating
Holstein cows in the first to third lactation and 10 to
75 days in milk.
Although there were no overt signs of mediator shock
during the first 5 days after vaccination, injection site
swelling was noted with each vaccine. The dairy was
equipped with a computerized system to monitor milk
production of each cow twice daily throughout lactation.
Some of the subjects experienced a decrease in milk pro-
duction compared with their baseline values (Table III).
Food Animal The Compendium January 1996

able (e.g., micromethod, mi-

TABLE I
crotechnique, microdilution,
Comparison of Endotoxin Units in Some Commercially Available Vaccines and LAL-bead). In perform-
Vaccine a Endotoxin Content (EU/ml)b ing the gel-clot assay, a small
UCD J5 Escherichia coli experimental core antigen ≤100 amount of LAL solution is
added to an equal volume of
Commercial J5 E. coli core antigen 1,825
sample or standard solution.
Salmonella core antigen 5,470
If a gel-clot is formed after
E. coli pilus vaccine 2,930,000
appropriate incubation, the
Lepto 5-way 52,500
assay is scored positive. The
Pasteurella hemolytica, P. multocida, 870,400 turbidimetric method is
Salmonella typhimurium an extension of the gel-clot
Haemophilus somnus 117,000 system. The turbidimetric
BRC, Clostridium perfringens, E. coli 38,800 reagent contains enough co-
S. typhimurium 2,975 agulogen to form a turbid so-
S. dublin/typhimurium bacterin 33,875 lution (not a gel-clot) when
Campylobacter fetus, Lepto 5-way 155,000 cleaved by clotting enzyme.
P. hemolytica 97,200 The amount of turbidity
IBR, PI3, H. somnus, P. hemolytica, P. multocida 226,500 formed is proportional to the
C. fetus, H. somnus, Lepto 5-way 49,950 amount of active clotting en-
H. somnus, Lepto 5-way 414,250 zyme and is thus proportional
Eight species of Clostridium 10.1 to the amount of endotoxin
C. perfringins, types C and D 0.51 present in the test solution.
IBR, BVD, PI3, Lepto 5-way 96,575 The colorimetric method
IBR, BVD, PI3 183,100 requires the mixing and in-
IBR, BVD, PI3, BRSV 143,000 cubation of test sample and
IBR, BVD, PI3, BRSV (live virus) 2.4 reagent. A precipitate that is
IBR, BVD, PI3, BRSV (killed virus) c
3.9 formed consists of turbid gel-
IBR, BVD, PI3, BRSV (killed virus) c
11,500 clot material. The gel-clot pre-
cipitate is centrifuged, collect-
a The Limulus amebocyte lysate (LAL) values may vary from lot to lot of the vaccine preparation.
b Endotoxin levels determined via LAL methodology by Associates of Cape Cod, Inc., Woods
ed, washed, and assayed by
the Lowry protein procedure.
Hole, MA.
Abbreviations: UCD = University of California-Davis, BRC = bovine respiratory complex, IBR = The amount of protein in the
infectious bovine rhinotracheitis, PI = parainfluenza, BVD = bovine virus diarrhea, BRSV = precipitate is directly propor-
bovine rhinotracheitis syncytial virus. tional to the amount of coag-
c Products from different manufacturers.
ulogen cleaved by the active
clotting enzyme. A standard
This decrease ranged from 2 to 8 pounds per milking for curve can be constructed to determine the endotoxin
at least 48 hours after the vaccines were administered. concentration in the sample.
There were no statistical increases in milk somatic cell The chromogenic method is similar to the turbidimet-
counts for the group during the 5-day observation period. ric assay system in that it is quantitative. The coagulogen
is partially or completely replaced by a chromogenic sub-
THE LIMULUS ASSAY strate. In a two-step method, the sample and LAL
Many LAL assay methods are available for assessing reagent are incubated and the chromogenic substrate is
the endotoxin content of body fluids, research reagents, then added to the mixture. After incubation, the reaction
pharmaceutical materials, and vaccine contents. These is halted by the addition of an acid solution. The com-
assays depend on the ability of endotoxin to coagulate a pleted reaction can be read via spectrophotometer.
protein isolated from the circulating amebocytes of the The formats of LAL assays vary among manufacturers
horseshoe crab Limulus polyphemus. At least four central because of the combinations of constituents and relative
methods of the LAL are used in the endotoxin-testing amounts of components in each product. There are many
arena: the gel-clot, turbidimetric (spectrophometric), versions of this test system; this brief discussion does not
colorimetric (Lowry protein), and chromogenic assays. describe all endotoxin assays currently on the market.
Several modifications of the gel-clot method are avail- In controlled conditions, the Limulus assay can be

MILK SOMATIC CELL COUNTS ■ ASSAY METHODS ■ GEL-CLOT SYSTEM

The Compendium January 1996 Food Animal

used for direct detection of vaccines is defined as the

TABLE II
contaminating endotoxins “freedom from properties
in many products and bio- Vaccine Immunogens and causing undue local or sys-
logic fluids. Some cyto- Endotoxin Units per Milliliter temic reactions when used
kines, such as tumor Endotoxin as recommended or suggest-
necrosis factor, may syner- Content ed by the manufacturer.”
gize with contaminating Vaccine Immunogens (EU/ml) In the same code, unfavor-
endotoxins and other mi- A Pasteurella hemolytica, 102,000 able reactions are defined
crobial products at levels P. multocida, Salmonella as “overt adverse changes
that cannot be reliably de- typhimurium which occur in healthy test
tected by the Limulus as- animals subsequent to initi-
say. 36 A negative Limulus B Salmonella core antigen 5,470 ation of a test and manifest-
assay thus is not sufficient ed during the observation
evidence that endotoxins C Infectious bovine 469,000 period prescribed in the test
are unrelated to the clinical rhinotracheitis, parainfluenza 3 , protocol which are at-
bovine virus diarrhea, bovine
phenomenon being ob- tributable either to the bio-
rhinotracheitis syncytial virus
served. Other cell wall logical product being tested
products (e.g., peptido- or to factors unrelated to
glycans, protein toxins, fungal such product as determined by
polyglycans, and mycoplasmal li- TABLE III the responsible individual con-
poglycans) are also capable of ducting the test.” The experiment
Cows with Milk Loss for at Least
efficiently inducing the cytokine reported here indicates that, in
48 Hours After Vaccination
production associated with clini- clinical signs and milk produc-
cal mediator shock. 36 Trial Number of tion, vaccine administration pro-
Peptidoglycans are potent acti- Number Vaccine Cows duced undue local and systemic
vators of cytokines and are found 1 A 2 of 5 reactions in certain individuals.
in all bacterial cell walls.37 These The observations noted in this
2 B 1 of 5
cell wall products may be present field demonstration indicate that
in vaccines, biologic fluids, or 3 C 2 of 5 rigorous experimental investi-
commercial products and are not 4 Combination 2 of 5 gations should be designed to
specifically detected by the of 3 vaccines reevaluate the safety of food ani-
Limulus assay. mal vaccine preparations.
In spite of mandated safety
CONCLUSION considerations in the manufacture of current food ani-
In food animals, gram-negative bacteria are responsi- mal immunogens, deaths and illnesses related to vac-
ble for clinical conditions that range from simple diar- cination occur daily. The new emphasis in food safety
rhea to life-threatening meningoencephalitis. Current regarding pathogen reduction and chemical residue
treatments and management practices are only moder- avoidance should dictate that immunogens that create
ately successful; a better understanding of the mole- abnormalities in host defense or production parameters
cules responsible for the pathophysiologic changes asso- must be identified. Although the endotoxin content of
ciated with these disease processes is necessary. Various a vaccine is only one of several risk factors in adverse re-
experiments and clinical reports27 have supplied infor- actions, attempts should be made to reduce the amount
mation necessary to begin the process of studying the of endotoxin in vaccine preparations using currently
mediator-induced shock that develops during gram- available production technology. Veterinarians should
negative bacterial disease. Because of animal health and routinely reevaluate immunization protocols to mini-
well-being and food safety issues, the ability to deliver mize the exposure of animals to endotoxin.
cost-effective treatment in patients with gram-negative
bacterial disease and mediator-induced shock is ex- About the Authors
tremely important in food animal agriculture. In addi- Dr. Cullor and Mr. Smith are affiliated with the Dairy
tion, there must be continued efforts to prevent gram- Food Safety Laboratory, Department of Pathology,
negative disease via new immunogens and improved Microbiology, and Immunology, College of Veterinary
management practices. Medicine, University of California, Davis, California.
In the Code of Federal Regulations (9 CFR), safety in

COLORIMETRIC METHOD ■ CHROMOGENIC METHOD ■ MENINGOENCEPHALITIS

Food Animal
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