Endotoxin And Disease In Food Animals

Vol.18, No. 1

January 1996

V

Continuing Education Article

FOCAL POINT ★ Conditions ranging from diarrhea to life-threatening meningoencephalitis are caused by gram-negative bacteria in food animals; current treatments must be improved by an enhanced understanding of the molecules responsible for the pathophysiologic changes. ■

KEY FACTS ■ A detailed description of the endotoxin complex includes portions of the cell membrane, cell envelope, lipoprotein, outer membrane, lipopolysaccharide, bacteria capsule, and glycocalyx. ■ Diseases in food animals that are associated with gram-negative organisms include neonatal coliform septicemia, coliform mastitis, salmonellosis, and pneumonia. ■ Morbidity and mortality associated with gram-negative bacterial sepsis, in part, are a result of host reactions to bacterial cell wall components. ■ Veterinarians should routinely reevaluate immunization protocols to minimize the exposure of food animals to endotoxin.

Endotoxin and Disease in Food Animals* University of California

James S. Cullor, DVM, PhD W. L. Smith, BS, MT

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ram-negative bacteria are responsible for many clinical conditions observed in food animal species. Current treatments and management practices are only moderately successful in ameliorating the clinical consequences of gram-negative infections. Future research and clinical approaches to managing these devastating diseases depend on a better understanding of the origin of endotoxin and how various cells and organ systems react to its presence in the body. This article considers clinically relevant information regarding the sources, structure, and biologic activities of endotoxin.

COMPONENTS OF THE GRAM-NEGATIVE BACTERIAL ENDOTOXIN COMPLEX Endotoxins have been demonstrated to be localized on the surface of bacterial cells and to form (together with phospholipids and proteins) the outer membrane of gram-negative bacteria (Figure 1). A detailed description of the endotoxin complex should include various portions of the cell membrane, the cell envelope, lipoprotein, outer membrane, lipopolysaccharide (LPS), bacteria capsule, and glycocalyx.1 The cell envelope is composed of the macromolecular layers that surround the bacterium. In gram-negative bacteria, the envelope includes a cell membrane, a cell wall, and possibly a capsule and/or a glycocalyx layer. Gram-negative bacteria possess a typical cell membrane that is composed of phospholipids and proteins. The structure contains the cytochromes and enzymes involved in electron transport and oxidative phosphorylation. This portion of the bacteria also contains chemoreceptors and is the site of action of certain antibiotics (e.g., polymyxin). The cell wall is the portion of the cell envelope that is external to the cytoplasmic membrane and internal to the capsule or glycocalyx. In gramnegative bacteria, the cell wall is composed of peptidoglycan, lipoprotein, the outer phospholipid membrane, and lipopolysaccharide. This structure gives the organism osmotic protection and gram-staining characteristics. *Supported in part by USDA formula funds, the Livestock Disease Research Laboratory, and the California Milk Advisory Board. All funds administered by the School of Veterinary Medicine, University of California, Davis, California.

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Peptidoglycan (also renary pili are involved in ferred to as mucopeptide or bacterial adherence. murein) is found in all bacLIPID-A terial cell walls. It is a comLipopolysaccharides conplex polymer that consists stitute the O antigens and of a backbone and a set of endotoxins of gram-negaidentical tetrapeptide side tive bacteria. The structure chains. Peptidoglycan is the of lipid-A from various bacsite of action of certain anteria has been described tibiotics, including peniand chemically synthecillin and cephalosporins. sized.2–4 Through this proLipoprotein provides a cess, some of the intrinsic strong cross-link between heterogeneity of the lipid-A the peptidoglycan and outmolecule has been deterer membrane. The outer mined to be related to the membrane is a phospholipid bilayer in which the Figure 1—Schematic representation of the components of presence of partial structures resulting from incomphospholipids of the outer gram-negative bacteria (LPS = lipopolysaccharide). plete synthesis. Lipid-A portion are replaced by constitutes the covalently lipopolysaccharides. This linked lipid component of lipopolysaccharide and is the structure is responsible for protecting the cells from endotoxic moiety of gram-negative bacteria. harmful enzymes and preventing leakage of periplasmic The polysaccharide and lipid portions of lipopolyproteins. saccharide contribute to the pathogenic potential of this Lipopolysaccharide is found in gram-negative bacteclass of bacteria by forming the endotoxin complex that ria and consists of lipid-A (several long-chain fatty acids provides the mediator shock potential for the disease proattached to phosphorylated glucosamine disaccharide cess. The interaction of the lipid-A molecule with various units), a polysaccharide composed of a core and termicell types may elicit the secretion of bioactive medianal repeating units, and major surface antigens (includtors—such as tumor necrosis factor (TNF), interleukin-1 ing the O antigen, found in the polysaccharide compo(IL-1), and interleukin-6 (IL-6)—which can lead to sysnent). The lipopolysaccharide is also referred to as temic mediator shock events. endotoxin, and the lipid-A portion is prominently associated with bacterial toxicity. GRAM-NEGATIVE BACTERIAL DISEASE The capsule is a well-defined structure of polysacchaGram-negative organisms are responsible for many ride that surrounds the bacterial cell and is generally lodiseases in food animals, including neonatal coliform cated external to the cell wall. The structure contributes septicemia, coliform mastitis, salmonellosis, pneumoto bacterial invasiveness because it provides bacterial renias caused by Pasteurella and Actinobacillus species, sistance to phagocytosis. brucellosis, metritis, campylobacteriosis, infections of The term glycocalyx refers to a loose network of polysaccharide fibrils that surrounds some bacterial cell the cornea and sclera, and thromboembolic meningoenwalls; the glycocalyx is sometimes referred to as the cephalitis. Epidemiologic studies in food animal neoslime layer of the bacteria. It is associated with many nates (e.g., cattle, pigs, and small ruminants) demonadhesive properties of the bacterial cell and contains strate that most clinical infections and mortality in this prominent antigenic sites. age group result from gram-negative organisms, specifiTwo other commonly mentioned portions of gramcally Escherichia coli and Salmonella species.5–10 Mortality varies among farms and production units; estimates negative bacteria that are not considered a part of the of dairy calf death losses during the first eight weeks of endotoxin complex are the flagella and pili (fimbriae). life range from 2.5% to 29%.11 Flagella are protein appendages used by the bacteria for Septicemia caused by E. coli (colisepticemia) generally locomotion. They may cover the entire bacterial cell affects calves younger than 1 week of age and is characsurface or be located only in one area of the cell. The terized by a rapidly fatal disease course. Low serum imflagella are composed of a protein subunit (called flagmunoglobulin concentration and exposure to invasive ellin) that may provide many antigenic determinants. serotypes of E. coli are major determinants of the develThe pili are rigid surface appendages primarily made of opment of colisepticemia.7,12–15 Death losses from salmoprotein molecules with antigenic properties. The ordiLIPID-A ■ MAJOR SURFACE ANTIGENS ■ TUMOR NECROSIS FACTOR ■ SEPTICEMIA

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nellosis in cattle are most severe in confinement-raised dairy calves at 1 to 10 weeks of age.9,10 Although salmonellosis is usually described as being confined to the gastrointestinal 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) occurs 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 bacteria 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, including initiation of complement and coagulation cascades and recruitment of activated neutrophils and macrophages. Pasteurella hemolytica produces a protein cytotoxin that is lethal to these macrophages and neutrophils; enzymes that can destroy tissue are thus released into the microenvironment. In addition, reactive 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, Enterobacter aerogenes, and Klebsiella pneumoniae. Other gram-negative organisms that are less commonly isolated include Pseudomonas aeruginosa, P. multocida, and Serratia marcescens. The resultant bacterial growth in the mammary gland can cause serious local and systemic consequences.18–21

CLINICAL SIGNS ASSOCIATED WITH DISEASE: MEDIATOR SHOCK Morbidity and mortality associated with gram-negative bacterial sepsis are apparently the consequences of host reaction to bacterial cell wall components (e.g., endotoxin, 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) endotoxin from gram-negative bacteria; (2) peptidoglycan and exotoxins from gram-negative bacteria; (3) endotoxin-binding proteins and receptors; (4) bactericidal pro-

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teases; (5) release of cytokines, histamine, bradykinin, and arachidonic acid metabolites; (6) complement activation; and (7) endothelium-derived adhesion molecules. The arachidonic acid metabolites originate in the cyclooxygenase 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 lipoxygenase pathway (leukotrienes) include 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotrienes (LT) A4, B4, C4, and D4. These compounds are potent bronchoconstrictors and vasoconstrictors, elicit plasma exudation, 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, polysaccharide 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 purified 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 inflammatory 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, transitory 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 reactions are lymphopenia followed by lymphocytosis, neutropenia followed by leukocytosis, hyperglycemia followed 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 intravascular coagulation with bilateral renal cortical necrosis), 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

SHIPPING FEVER PNEUMONIA ■ COLIFORM MASTITIS ■ CYCLOOXYGENASE PATHWAY

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Effects of Endotoxins General ■ Lethargy ■ Respiratory distress ■ Transitory hyperthermia followed by hypothermia ■ Decreased systemic blood pressure ■ Increased heart rate followed by decreased cardiac output ■ Diarrhea ■ Changes in blood cell counts ■ Alterations in the blood coagulation system Specific ■ Lymphopenia followed by lymphocytosis ■ Neutropenia followed by leukocytosis ■ Hyperglycemia followed by hypoglycemia ■ Depletion of liver glycogen ■ Anabolic and catabolic responses in protein metabolism ■ Schwartzman reactions ■ Transitory tolerance to further endotoxin insults ■ Altered reproductive performance

and extrinsic pathways of the clotting system also are activated, expression of tissue factor is enhanced, and disseminated intravascular coagulation may ensue. Platelets aggregate and sequester in various capillary locations and secrete their mediators. Neutrophils respond by producing inflammatory mediators (prostaglandins or leukotrienes) and oxygen radicals. Macrophage function is enhanced, and the cells secrete cytokines. Comprehensive discussions of the pathophysiologic effects of endotoxins in ruminants are available in the literature.26,27

ANTIBIOTIC THERAPY AND ENDOTOXIN RELEASE Antibiotic-induced release of endotoxin has been of clinical and research interest for some time. A 3- to 78fold increase in the total concentration of endotoxin in vitro and in vivo has been reported. 28,29 There is apparently considerable overlap between the effect of β-lactam antibiotics and non–βlactam antibiotics, with an unexplained delay between the lethal activity of antibiotics and the release of endotoxin. The lytic and nonlytic release of endotoxin thus must be considered in the pathogenesis of disease and will influence the therapeutic efficacy of antiendotoxin 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

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 classical clinical signs mentioned. Although it is a strong pyrogen in the host, endotoxin is weakly toxic, rarely fatal compared with exotoxins, and a relatively poor immunogen. No toxoid preparation, in the classical scientific definition, is provided in most vaccine preparations of this molecule. The appropriate definition of a toxoid dictates that the endotoxin molecule in the preparation be treated 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, enterotoxin, or neurotoxin) with defined actions on cells or tissue. Exotoxins are highly toxic and often result in a fatal disease process. Compared with endotoxins, these bacterial proteins are highly immunogenic and stimulate the production of neutralizing antibody (antitoxin). Treating the protein toxin with formaldehyde eliminates its toxicity 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 permeability that lead to diarrhea. The substantial diarrhea observed in patients with cholera is due to the action of this type of toxin and is commonly caused by foodpoisoning microorganisms.

ESCHERICHIA COLI 0157:H7 AND VEROTOXINS During the winter of 1993, a severe outbreak of foodborne 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 locations, and the number of cases reached 400. Of these, 125 patients were hospitalized. At least 29 patients developed acute renal failure, and all but 8 of these required hemodialysis. Three young children died. Although this outbreak received deserved public attention, it is not unique. Escherichia coli 0157:H7 was first identified in 1982, when it was determined to be the cause of a multistate outbreak of hemorrhagic colitis associated 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 municipal water supplies.

ANTIBIOTIC-INDUCED RELEASE ■ ANTIENDOTOXIN THERAPY ■ HEAT-LABILE PROTEINS

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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, antibiotics, 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 resulted in the following classifications: enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), and verotoxigenic (VTEC).32 Canadian investigators have demonstrated that toxins produced by strains of E. coli serotype 0157:H7 are cytotoxic for vero cells; hence the term verotoxins. In addition, isolation of the pathogen was closely related with hemorrhagic 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 constituted by an A (active) subunit and several B (binding) subunits. The verotoxins bind to the receptor on the surface of an intestinal cell via the B subunit. The A subunit is then taken into the cell and cleaved to an active fragment 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 culture-positive feces were found among 633 calves sampled during the first week of life. Culture-positive calves were

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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 veterinarians and producers. The Limulus amebocyte lysate (LAL) test was used to determine endotoxin levels (endotoxin units [EU/ml]) present in commercial vaccine preparations. The usual conversion of 5 EU/ng of endotoxin applies 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 target amount in a 700-kg cow would be 3500 EU. No pyrogenic thresholds have been established for food animals related to vaccine administration. Many of the immunization 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 administered. For example, the veterinarian may need to have the products tested and then weigh safety and efficacy considerations in selecting the immunogen to be administered 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 manufacturer 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 production compared with their baseline values (Table III).

CONTAMINATED MEAT ■ VEROTOXINS ■ CULTURE-POSITIVE FECES ■ IMMUNOGENS

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able (e.g., micromethod, microtechnique, microdilution, and LAL-bead). In performing the gel-clot assay, a small Vaccine a Endotoxin Content (EU/ml)b amount of LAL solution is UCD J5 Escherichia coli experimental core antigen ≤100 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 coS. typhimurium 2,975 agulogen to form a turbid soS. 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 enzyme and is thus proportional H. somnus, Lepto 5-way 414,250 to the amount of endotoxin Eight species of Clostridium 10.1 present in the test solution. C. perfringins, types C and D 0.51 The colorimetric method IBR, BVD, PI3, Lepto 5-way 96,575 requires the mixing and inIBR, BVD, PI3 183,100 cubation of test sample and IBR, BVD, PI3, BRSV 143,000 reagent. A precipitate that is IBR, BVD, PI3, BRSV (live virus) 2.4 formed consists of turbid gelc IBR, BVD, PI3, BRSV (killed virus) 3.9 clot material. The gel-clot prec IBR, BVD, PI3, BRSV (killed virus) 11,500 cipitate is centrifuged, collecta The Limulus amebocyte lysate (LAL) values may vary from lot to lot of the vaccine preparation. ed, washed, and assayed by b Endotoxin levels determined via LAL methodology by Associates of Cape Cod, Inc., Woods the Lowry protein procedure. Hole, MA. The amount of protein in the Abbreviations: UCD = University of California-Davis, BRC = bovine respiratory complex, IBR = precipitate is directly proporinfectious bovine rhinotracheitis, PI = parainfluenza, BVD = bovine virus diarrhea, BRSV = tional to the amount of coagbovine rhinotracheitis syncytial virus. 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 turbidimetcounts 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 subTHE 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 comassays 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 availIn controlled conditions, the Limulus assay can be TABLE I Comparison of Endotoxin Units in Some Commercially Available Vaccines

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

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used for direct detection of TABLE II contaminating endotoxins Vaccine Immunogens and in many products and bioEndotoxin Units per Milliliter logic fluids. Some cytokines, such as tumor Endotoxin Content necrosis factor, may synerVaccine Immunogens (EU/ml) gize with contaminating endotoxins and other miA Pasteurella hemolytica, 102,000 crobial products at levels P. multocida, Salmonella that cannot be reliably detyphimurium tected by the Limulus asB Salmonella core antigen 5,470 say. 36 A negative Limulus assay thus is not sufficient C Infectious bovine 469,000 evidence that endotoxins rhinotracheitis, parainfluenza , 3 are unrelated to the clinical bovine virus diarrhea, bovine phenomenon being obrhinotracheitis syncytial virus served. Other cell wall products (e.g., peptidoglycans, protein toxins, fungal polyglycans, and mycoplasmal liTABLE III poglycans) are also capable of Cows with Milk Loss for at Least efficiently inducing the cytokine 48 Hours After Vaccination production associated with clini36 Trial Number of cal mediator shock. Number Vaccine Cows Peptidoglycans are potent activators of cytokines and are found 1 A 2 of 5 in all bacterial cell walls.37 These 2 B 1 of 5 cell wall products may be present 3 C 2 of 5 in vaccines, biologic fluids, or commercial products and are not 4 Combination 2 of 5 specifically detected by the of 3 vaccines Limulus assay.

CONCLUSION In food animals, gram-negative bacteria are responsible for clinical conditions that range from simple diarrhea to life-threatening meningoencephalitis. Current treatments and management practices are only moderately successful; a better understanding of the molecules responsible for the pathophysiologic changes associated with these disease processes is necessary. Various experiments and clinical reports27 have supplied information necessary to begin the process of studying the mediator-induced shock that develops during gramnegative bacterial disease. Because of animal health and well-being and food safety issues, the ability to deliver cost-effective treatment in patients with gram-negative bacterial disease and mediator-induced shock is extremely important in food animal agriculture. In addition, there must be continued efforts to prevent gramnegative disease via new immunogens and improved management practices. In the Code of Federal Regulations (9 CFR), safety in

vaccines is defined as the “freedom from properties causing undue local or systemic reactions when used as recommended or suggested by the manufacturer.” In the same code, unfavorable reactions are defined as “overt adverse changes which occur in healthy test animals subsequent to initiation of a test and manifested during the observation period prescribed in the test protocol which are attributable either to the biological product being tested or to factors unrelated to such product as determined by the responsible individual conducting the test.” The experiment reported here indicates that, in clinical signs and milk production, vaccine administration produced undue local and systemic reactions in certain individuals. The observations noted in this field demonstration indicate that rigorous experimental investigations should be designed to reevaluate the safety of food animal vaccine preparations. In spite of mandated safety considerations in the manufacture of current food animal immunogens, deaths and illnesses related to vaccination occur daily. The new emphasis in food safety regarding pathogen reduction and chemical residue avoidance should dictate that immunogens that create abnormalities in host defense or production parameters must be identified. Although the endotoxin content of a vaccine is only one of several risk factors in adverse reactions, attempts should be made to reduce the amount of endotoxin in vaccine preparations using currently available production technology. Veterinarians should routinely reevaluate immunization protocols to minimize the exposure of animals to endotoxin.

About the Authors Dr. Cullor and Mr. Smith are affiliated with the Dairy Food Safety Laboratory, Department of Pathology, Microbiology, and Immunology, College of Veterinary Medicine, University of California, Davis, California.

COLORIMETRIC METHOD ■ CHROMOGENIC METHOD ■ MENINGOENCEPHALITIS

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