Professional Documents
Culture Documents
1 January 1996 V
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 The Compendium January 1996
nellosis in cattle are most severe in confinement-raised teases; (5) release of cytokines, histamine, bradykinin,
dairy calves at 1 to 10 weeks of age.9,10 Although salmo- and arachidonic acid metabolites; (6) complement acti-
nellosis is usually described as being confined to the gas- vation; and (7) endothelium-derived adhesion molecules.
trointestinal tract, most calves develop bacteremia with The arachidonic acid metabolites originate in the cy-
spread of the infection to liver, lungs, bone marrow, and clooxygenase and lipoxygenase pathways. Members of
central nervous system. the cyclooxygenase pathway and their biologic activities
In older cattle, shipping fever pneumonia (SFP) oc- include PGE2 (vasodilator), PGF2 (vasoconstrictor),
curs in feedlot animals; 75% of cases develop during thromboxane A 2 (vasoconstrictor and promoter
the first 45 days that the cattle are housed at the feedlot of platelet aggregation), and PGI 2 (vasodilator and
facility. The disease process is apparently caused by a inhibitor of platelet aggregation). Members of the lipoxy-
complex interaction among stressors, viruses, and bac- genase pathway (leukotrienes) include 5-hydroxyeico-
teria that have an endotoxin component in the cell satetraenoic acid (5-HETE) and leukotrienes (LT) A4,
wall. The primary bacterial isolates of shipping fever B4, C4, and D4. These compounds are potent bron-
pneumonia are Pasteurella hemolytica, P. multocida, choconstrictors and vasoconstrictors, elicit plasma exuda-
and some Pseudomonas species.16 The capsules of P. tion, are chemotactic for leukocytes, and are involved in
hemolytica, P. multocida, and Haemophilus somnus microthrombus formation.
contain lipopolysaccharide (endotoxin). The release of Variations in the biologic activity of endotoxins have
the endotoxin molecule induces several events, includ- been observed. These differences relate to the presence
ing initiation of complement and coagulation cascades of lipid-A–associated protein, aggregation, polysaccha-
and recruitment of activated neutrophils and macro- ride composition, culture conditions, and the source of
phages. Pasteurella hemolytica produces a protein cyto- the organism. Many of the clinical signs observed in
toxin that is lethal to these macrophages and neu- conjunction with gram-negative bacterial disease have
trophils; enzymes that can destroy tissue are thus been reproduced experimentally by administering puri-
released into the microenvironment. In addition, reac- fied endotoxin (also known as lipopolysaccharide) in
tive oxygen intermediates are produced that are capable various doses and by various routes. The effects of
of destroying neutrophils and surrounding tissue. lipopolysaccharide on host cells (e.g., macrophages,
Coliform mastitis, another disease process initiated platelets, and endothelial cells) and on the release of in-
by gram-negative opportunists, can be devastating to flammatory mediators also influence the clinical signs
dairy production units. The process, which primarily observed at various stages of the disease process. For
develops during the first 100 days of lactation but may example, the severity of inflammatory responses after
occur at any stage of lactation or during the dry period, lipopolysaccharide challenge has been demonstrated to
encompasses all degrees of severity from peracute to vary directly with receptor numbers on the macrophage
subclinical. 17 The bacteria most frequently isolated (which can vary among animals).25
from this form of bovine mastitis include E. coli, En- The general effects of endotoxins are well chronicled
terobacter aerogenes, and Klebsiella pneumoniae. Other and reportedly include lethargy, respiratory distress, tran-
gram-negative organisms that are less commonly isolat- sitory hyperthermia followed by hypothermia, decreased
ed include Pseudomonas aeruginosa, P. multocida, and systemic blood pressure, increased heart rate followed by
Serratia marcescens. The resultant bacterial growth in decreased cardiac output, diarrhea, changes in blood cell
the mammary gland can cause serious local and sys- counts, and alterations in the blood coagulation system.
temic consequences.18–21 Some of the more specific physiologic and pathologic re-
actions are lymphopenia followed by lymphocytosis, neu-
CLINICAL SIGNS ASSOCIATED tropenia followed by leukocytosis, hyperglycemia fol-
WITH DISEASE: MEDIATOR SHOCK lowed by hypoglycemia, depletion of liver glycogen,
Morbidity and mortality associated with gram-nega- anabolic and catabolic responses in protein metabolism,
tive bacterial sepsis are apparently the consequences of localized and generalized Shwartzman reactions (i.e., local
host reaction to bacterial cell wall components (e.g., en- thrombosis formation and/or general disseminated in-
dotoxin, the lipopolysaccharide cell wall component of travascular coagulation with bilateral renal cortical necro-
gram-negative bacteria).22–24 The following endogenous sis), induction of transitory tolerance to further endotoxin
and exogenous factors, however, have been linked to the insults, and altered reproductive performance (see the
pathophysiology of sepsis and mediator shock: (1) endo- box).
toxin from gram-negative bacteria; (2) peptidoglycan The classical and alternative complement pathways are
and exotoxins from gram-negative bacteria; (3) endotox- activated, resulting in generation of anaphylatoxin and
in-binding proteins and receptors; (4) bactericidal pro- many secondary local and systemic effects. The intrinsic
and extrinsic pathways of the growth and bacterial lysis as part of the outer membrane
Effects of clotting system also are acti- of gram-negative bacteria. The release of this compound
Endotoxins vated, expression of tissue results in the initiation of mediator cascades (e.g., release
factor is enhanced, and dis- of cytokines, serotonin, histamine, bradykinin, and
General seminated intravascular coag- arachidonic acid metabolites) that culminate in the clas-
ulation may ensue. Platelets sical clinical signs mentioned. Although it is a strong py-
■ Lethargy
aggregate and sequester in rogen in the host, endotoxin is weakly toxic, rarely fatal
■ Respiratory distress various capillary locations and compared with exotoxins, and a relatively poor immuno-
■ Transitory secrete their mediators. Neu- gen. No toxoid preparation, in the classical scientific def-
hyperthermia followed trophils respond by produc- inition, is provided in most vaccine preparations of this
by hypothermia ing inflammatory mediators molecule. The appropriate definition of a toxoid dictates
■ Decreased systemic (prostaglandins or leuko- that the endotoxin molecule in the preparation be treat-
trienes) and oxygen radicals. ed by chemicals or heat in such a way as to eliminate the
blood pressure
Macrophage function is en- toxic qualities while retaining the antigenic properties.
■ Increased heart rate hanced, and the cells secrete These data are often unavailable to the public.
followed by decreased cytokines. Comprehensive Exotoxins are heat-labile proteins excreted by certain
cardiac output discussions of the pathophysi- gram-positive or gram-negative bacteria. The molecules
■ Diarrhea ologic effects of endotoxins in possess a specific mode of action (e.g., cytotoxin, entero-
■ Changes in blood cell ruminants are available in the toxin, or neurotoxin) with defined actions on cells or tis-
literature.26,27 sue. Exotoxins are highly toxic and often result in a fatal
counts
disease process. Compared with endotoxins, these bacte-
■ Alterations in the blood ANTIBIOTIC THERAPY rial proteins are highly immunogenic and stimulate the
coagulation system AND ENDOTOXIN production of neutralizing antibody (antitoxin). Treating
RELEASE the protein toxin with formaldehyde eliminates its toxici-
Specific Antibiotic-induced release ty without destroying the immunogenic properties.
■ Lymphopenia followed of endotoxin has been of Formaldehyde treatment of endotoxin does not make the
clinical and research interest lipopolysaccharide molecule a toxoid as strictly defined.
by lymphocytosis
for some time. A 3- to 78- Exotoxins reportedly do not produce fever in the host.
■ Neutropenia followed fold increase in the total con- Enterotoxins are exotoxins that specifically affect the
by leukocytosis centration of endotoxin in small intestine, causing changes in intestinal permeabil-
■ Hyperglycemia vitro and in vivo has been re- ity that lead to diarrhea. The substantial diarrhea ob-
followed by ported. 28,29 There is appar- served in patients with cholera is due to the action of
hypoglycemia ently considerable overlap this type of toxin and is commonly caused by food-
between the effect of β-lac- poisoning microorganisms.
■ Depletion of liver
tam antibiotics and non–β-
glycogen lactam antibiotics, with an ESCHERICHIA COLI 0157:H7 AND VEROTOXINS
■ Anabolic and catabolic unexplained delay between During the winter of 1993, a severe outbreak of food-
responses in protein the lethal activity of antibi- borne human disease in the Pacific Northwest was
metabolism otics and the release of endo- linked to microbial contamination of ground beef with
■ Schwartzman reactions toxin. The lytic and nonlytic E. coli 0157:H7.30 The outbreak occurred in several lo-
release of endotoxin thus cations, and the number of cases reached 400. Of these,
■ Transitory tolerance to
must be considered in the 125 patients were hospitalized. At least 29 patients de-
further endotoxin pathogenesis of disease and veloped acute renal failure, and all but 8 of these re-
insults will influence the therapeutic quired hemodialysis. Three young children died. Al-
■ Altered reproductive efficacy of antiendotoxin though this outbreak received deserved public attention,
performance therapy. Scientific research is it is not unique. Escherichia coli 0157:H7 was first iden-
necessary concerning this tified in 1982, when it was determined to be the cause
topic in food animals. of a multistate outbreak of hemorrhagic colitis associat-
ed with hamburger patties sold by a national fast-food
ENDOTOXINS, EXOTOXINS, chain.31 Other recent outbreaks of E. coli 0157:H7–
AND ENTEROTOXINS related disease have been associated with contaminated
Endotoxins are heat-stable lipopolysaccharide– apple cider, unpasteurized milk, mayonnaise, and mu-
lipoprotein complexes that may be released during cell nicipal water supplies.
Because some of the most significant clinical events present in all regions of the United States; the herd
have involved contaminated meat, the public view of prevalence was estimated at 5%.35 No information was
food-borne disease and the safety standards required to reported concerning the capability of these isolates to
prevent its occurrence has changed. Veterinarians should produce verotoxins.
be active in improving on-farm, preharvest, food safety
quality assurance programs to prevent contamination of VACCINES AND ENDOTOXIN CONTENT
milk and meat by pesticides, herbicides, hormones, an- Traditional gram-negative vaccine preparations have
tibiotics, and microbes. been plagued by problems of adverse reactions in the
The vast majority of strains of E. coli isolated from host species, thus earning the distrust of many veterinari-
feces are part of the normal intestinal flora. They play ans and producers. The Limulus amebocyte lysate (LAL)
an important role in maintaining optimum intestinal test was used to determine endotoxin levels (endotoxin
physiology. In this group of bacteria, however, are units [EU/ml]) present in commercial vaccine prepara-
strains that are pathogenic and cause diarrhea. Strains tions. The usual conversion of 5 EU/ng of endotoxin ap-
that cause diarrhea do so by mechanisms that have re- plies to all of the figures in this report. Commercial
sulted in the following classifications: enteropathogenic gram-negative immunogens contain thousands of
(EPEC), enterotoxigenic (ETEC), enteroinvasive EU/ml of vaccine and may contain millions of EU/ml of
(EIEC), and verotoxigenic (VTEC).32 Canadian inves- free endotoxin, as measured by the LAL (Table I).
tigators have demonstrated that toxins produced by Because the pyrogenic threshold for pharmaceutical
strains of E. coli serotype 0157:H7 are cytotoxic for compounds is 5 EU/kg body weight, the maximum tar-
vero cells; hence the term verotoxins. In addition, isola- get amount in a 700-kg cow would be 3500 EU. No py-
tion of the pathogen was closely related with hemor- rogenic thresholds have been established for food animals
rhagic colitis (HC) syndrome in humans. related to vaccine administration. Many of the immu-
Two clinically important verotoxins produced by E. nization schedules used today in food animals may exceed
coli (VT1 and VT2) are members of a family of many the target amount set in the pharmaceutical compound
similar cytotoxins. The verotoxins are subunit toxins con- example. As producers become more aware of the adverse
stituted by an A (active) subunit and several B (binding) reactions that may result from immunization protocols,
subunits. The verotoxins bind to the receptor on the sur- the veterinarian in charge of herd health programs must
face of an intestinal cell via the B subunit. The A subunit be aware of the endotoxin levels in the vaccines being ad-
is then taken into the cell and cleaved to an active frag- ministered. For example, the veterinarian may need to
ment that inhibits cellular protein synthesis. Escherichia have the products tested and then weigh safety and effica-
coli 0157:H7 is often the most frequent serotype of cy considerations in selecting the immunogen to be ad-
VTEC isolated. The reported predominance of serotype ministered or the frequency of vaccine administration. To
0157 is undoubtedly biased by the wide use of methods date, no published research studies have assessed either of
adapted only for this serotype. More than 57 other these strategies using commercial vaccine preparations.
serotypes that produce verotoxins have been described.33
A German study used a common biotechnology EXPERIMENTAL FINDINGS
technique (DNA–DNA colony hybridization using An experiment of importance to the dairy industry
specific gene probes for VT1 and VT2) to examine involved the administration of commercially available
2100 E. coli strains from the feces of healthy animals. immunogens to lactating dairy cattle and the effect of
Ten of 82 milk cows, 20 of 212 beef cattle, and 5 of 75 this practice on milk production during the next few
pigs reportedly carried genes for VT1 and/or VT2. days (Table II). The immunogens were administered
Several of the serotyped isolates have been described to via the dose and route recommended by the manufac-
be pathogenic in humans (0157:H7, 082:H8, 0116, turer and were administered alone or in combination
0113, 0126, and 091).34 with the other two vaccines. The subjects were lactating
In a portion of the National Dairy Heifer Evaluation Holstein cows in the first to third lactation and 10 to
Project conducted by Veterinary Services (USDA/ 75 days in milk.
APHIS), 6894 heifer calves in 1068 dairy herds were Although there were no overt signs of mediator shock
sampled in 28 states. The study reported a prevalence of during the first 5 days after vaccination, injection site
isolation of E. coli 0157:H7 in calves of 3.6 per 1000. swelling was noted with each vaccine. The dairy was
Escherichia coli 0157:H7 was found among calves from equipped with a computerized system to monitor milk
2 weeks to greater than 12 weeks of age; however, no cul- production of each cow twice daily throughout lactation.
ture-positive feces were found among 633 calves sampled Some of the subjects experienced a decrease in milk pro-
during the first week of life. Culture-positive calves were duction compared with their baseline values (Table III).
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