Endotoxin, an extremely hydrophobic, heat- and acid-resistant molecule of lipid A, keto-octanoic acid, and complex sugars, makes up most of the cell wall of all aerobic and anaerobic Gram-negative bacteria (Figure 1). It is released when the cell wall is disrupted, which occurs when the cell dies or is Iysed by chemotherapeutic agents. Most endotoxin entering the body is rapidly eliminated by the liver, but when the host’s clearance mechanisms are overwhelmed, endotoxin reaches the systemic circulation. There, it rapidly affects the diseased host’s peripheral circulation, cardiovascular dynamics, thermal control mechanisms, coagulation mechanism, prostaglandin metabolism, complement cascade, immune system, and inflammatory process. The classic signs of endotoxemia are depression of the central nervous system, hyperpnea, dyspnea, anorexia, pyrexia, and leukopenia followed by leukocytosis.

Endotoxin is puzzling because the same milligram quantity required to kill a 20-g mouse will also kill a l,000-lb horse. However, bolus amounts are probably not seen in animals in practice; an insidious minuscule amount in the bloodstream of a bacteremic animal that exceeds the liver’s ability to detoxify is a more common finding. Minuscule amounts of endotoxin may gain entrance to the circulation system from an obscure abscess, a chronically infected urogenital tract, or damaged gut epithelium. After gaining entrance, endotoxin usually causes an endotoxic shock crisis with up to 50% mortality.

Lipid A, the toxic moiety of endotoxin, appears to be identical in all Gram-negative bacteria, though endotoxins from different Gram-negative bacteria exhibit slightly different toxicity’s. Differences are related to the biochemical arrangement and complexity of the sugars and side chains that compose the mucoid capsule of different Gram-negative bacteria. These sugars and side chains are covalently bonded to lipid A through keto-octanoic acid. The current serotyping system so deeply engrained in our psyches by our veterinary training automatically associates Escherichia coli serotype K99 with calf diarrhea, serotype typhimurium with equine salmonellosis, Pasteurella hemolytica serotype I with shipping fever complex, and a variety of E. coli serotypes with coliform mastitis. These bacteria, which cause endotoxin-associated diseases, are classified by sero-agglutination of their capsular materials (“O” side chains). However, these and most other Gram-negative bacteria have a common antigenic core (lipid A and keto-octanoic acid) in their cell walls. This common core offers the opportunity to genetically engineer workable cross-protective vaccines.2

Most of the vaccines currently available for Gram-negative bacterial diseases are made from several serotypes of the so-called smooth strains of bacteria. There are over 2,000 serotypes of Salmonella enteritidis alone, and an even greater number for E. coli and other bacteria. And new serotypes appear continually as a result of the exchange of genetic material among Gram-negative bacteria. It is not surprising, therefore, that the causative agents of Gram-negative diseases are always “shifting and drifting” and, more important, that currently available polyvalent vaccines are highly variable in eliciting protection for the host; vaccine breaks, or failures, are always lurking in the background. (Remember, though, that age, stress, previous exposures, current state of health, and the status of the immune system also influence a vaccine’s efficacy. The break is not always the fault of the vaccine.)

In the search for a “universal” vaccine, various laboratories have isolated or genetically engineered rough mutants of Gram-negative bacteria that have temporarily or permanently lost the ability to produce part (E. coli J-5)3,4 or all (S. typhimurium R 17)5 of their capsular or “O” side chain carbohydrates. Vaccines prepared with these rough mutants expose the bacterium’s usually protected naked core to the host’s immune system. Because this core is common to all Enterobacteriaceae and to most other Gram-negative bacteria, immunity elicited by these genetically engineered vaccines is cross-protective for essentially all Gram-negative bacterial diseases.

Inadequate colostral antibodies, inadequate colostral absorption, inability to suckle, and the presence of a primary immune deficiency disorder are all circumstances that may result in FPT or PFPT. Studies have shown that 19% to 24% of all foals experience FPT, which is indicated by serum levels of less than 200 mg/dl of lgG antibodies. Up to 75% of these foals require aggressive treatment of infections, and many of them will die. Foals with serum levels of 200 to 400 mg/dl of lgG antibodies are classified as PFPT foals. Twenty-five percent of these foals require aggressive treatment of acquired infections, and some will die.

FPT or PFPT in a neonate leaves it more vulnerable to pathogenic organisms in the environment. Although exposure to Escherichia coli and other microorganisms also predisposes the neonate to infection, septicemia in neonatal foals is probably related more to lgG antibody deficiency than to the virulence of a particular strain of microorganism. Unfortunately, in the first hours of life the intestinal epithelium is highly and non-selectively permeable: Bacteria and viruses can more freely cross the intestinal epithelial barrier, resulting in a high incidence of neonatal sepsis and mortality. Moreover, the danger of neonatal infection is worsened because the newborn requires 10 to 14 days to produce its own antibody response to any infectious agent. To help prevent infection, all newborn foals should receive 800 mg/dl of serum lgG antibodies immediately after birth.

Because FPT or PFPT is a predisposing factor in most foal deaths, it is prudent to measure total lgG antibody levels in foals during the neonatal period. One of the most convenient methods for measuring total lgG antibody serum levels is an enzymatic assay that provides results within a few minutes (Cite® Foal lgG–Idexx).

Banked, frozen colostrum is often used to supplement the mare’s colostrum. But extramaternal colostrum must be administered within 12 hours after birth to ensure that antibodies are absorbed. Serum lgG levels can be measured 24 hours after birth to determine the adequacy of antibodies provided by banked colostrum. If levels are inadequate at this time, sources of lgG antibodies other than colostrum are indicated. Other sources include Iyophilized lgG for oral administration, frozen plasma for intravenous administration, and refrigerated antiserum (see boxed text).

Studies of equine immunodeficiency and its role in increasing a foal’s vulnerability to deadly diseases suggest that immense economic losses result from inadequate immunization programs. It is obvious that much of this loss could be eliminated by employing the sound immunologic strategies now available.

EndoserumTM (IMMVAC), a recently developed antiserum containing high levels of anti-endotoxin antibodies, provides protection against most Gram-negative endotoxins, including those produced by E. coli and Salmonella species. This protection is possible because the epitope responsible for stimulating antibody production by the host’s immune system is the cell wall’s common-core antigen.

This common core is similar in virtually all Gram-negative organisms. Endoserum contains consistent USDA-regulated levels of cross-protective anti-core antigen (anti-endotoxin) antibodies as well as high lgG antibody levels. The concentration of total lgG in Endoserum is more than 3,000 mg/deciliter. The anti-endotoxin antibodies are especially advantageous for combating a variety of Gram-negative septicemia/endotoxemia problems in foals.

Normally, epithelial cells are produced in the intestinal crypts. As they mature, these cells migrate up the intestinal villi, eventually reaching the tips of the villi. There they produce digestive enzymes and absorb and transport essential nutrients for the host. Absorption, therefore, occurs at the villi’s tips, whereas most intestinal secretion is confined to goblet cells and immature epithelial cells lining the crypts. There are two mechanisms of bacterial infection: noninvasive and invasive. Most bacteria are noninvasive: They do not produce histopathologic changes in the mucosa or submucosa, and so inflammatory cells (fecal leukocytes) do not appear in the diarrheal stools. Noninvasive bacteria attach to the intestinal mucosa with specialized appendages, or pili. After pilus attachment, these bacteria replicate rapidly and secrete exotoxins that bind to receptors on the surfaces of the epithelial cells. These exotoxins work at the molecular level to stimulate adenyl cyclase to increase cyclic AMP. Cyclic AMP decreases absorption of sodium ions and increases the secretion of chloride ions across the cytoplasmic membrane of epithelial cells. This results in diarrhea–the loss of large quantities of water, as well as electrolytes, in the feces. Although these specific changes are not life threatening, the resulting dehydration predisposes the host to problems such as bovine and porcine enteric colibacillosis and the bacterial/endotoxin-mediated diarrheas associated with canine and feline parvoviral infections.

Invasive bacteria attach to the mucosa and colonize. But invasive bacteria invade epithelial cells and multiply intracellularly. During the first 24 to 48 hours of illness, these bacteria cause watery diarrhea, which becomes exudative and hemorrhagic (dysenteric) after the submucosa is penetrated. Intracellular invasion is rapidly followed by the depletion of iron and other essentiai nutrients. This depletion produces inflammatory damage to the underlying mucosa and the subsequent appearance of blood and leukocytes in stool specimens. Diseases resulting from this mechanism of bacterial infection include acute equine gastroenteritis caused by Salmonella typhimurium and swine dysentery caused by S. choleraesuis.

Diarrhea invariably alters the balance of the intestinal microflora. Impaired movement of luminal contents and increased water content of evacuated feces decrease numbers of lactobacilli and homofermentative streptococci. Accompanying this change is a concomitant increase in numbers of Enterobacteriaceae. The normal death of these increased numbers of Enterobacteriaceae increases endotoxin in the gut lumen. Endotoxin, aided by the damaged mucosal barrier and greater vascular permeability, can then enter the circulation. When endotoxemia complicates the diarrheal syndrome, it creates a potential for life-threatening irreversible hemorrhagic shock, disseminated intravascular coagulation, and acute oliguric renal failure.

Treatment of diarrhea is limited to fluid replacement and the appropriate use of nonsteroidal anti-inflammatory drugs, such as aspirin, which blocks cyclic AMP formation and mucosal secretion. To counter the electrolyte flux, replace fluids with natural products such as serum or plasma, with synthetic fluids such as lactated Ringer solution, or with products containing glucose and sodium.

Because antiserum treats specifically the pathophysiologic mechanism of diarrhea, products such as EndoserumTM (IMMVAC), which contains replacement electrolytes and cross-protective antibodies to enhance bacterial phagocytosis and neutralize endotoxin, also offer effective aid in treating diarrhea.

Historically, coliform mastitis, which is often accompanied by endotoxemia, has been treated with antimicrobials. Unfortunately, antimicrobial administration in lactating cows requires the disposal of milk during the administration period and during the required withdrawal time. Frequently, milk must be disposed of for two weeks. Treatment expenses and the loss of income due to milk disposal may cost the dairy owner more than $1,500 per case. What’s more antimicrobials may facilitate the persistence of antimicrobial-resistant Gram-negative serotypes and thereby increase the pool of resistant pathogens on a given dairy farm.

Vaccination, then, would seem to offer a better method for managing coliform mastitis.

ln dairy cows, coliform mastitis is most commonly associated with E. coli bacteria and endotoxins. Because there is no way of knowing in advance which specific serotype of a particular species of Gram-negative bacteria is responsible for any given case of coliform mastitis, it is impossible to formulate effective broad spectrum homologous vaccines. Such vaccines would need to contain numerous Gram-negative bacterins to provide any degree of cross-protection.

So a logical approach to formulating an efficacious vaccine would be to use a single antigen that induces the immune system to produce antibodies that cross- protect against Gram-negative organisms and their endotoxins. Specific R-mutants of a Salmonella sp and E. coli have been found to provide such cross-protection against septicemias and endotoxemias arising from various Gram-negative infections. The antibodies produced by these bacterins, which are coupled with a potent immune stimulant, have provided cross-protection to cows and horses either naturally challenged or arbitrarily challenged in the laboratory.

For example, in independent studies of California and Arizona dairy cows, mutant Cram-negative bacterins lowered the incidence and severity of coliform mastitis.

Endotoxemias are also associated with Gram-negative septicemias arising from E. coli and Salmonella diarrheas, and Pasteurella multocida and Pasteurella hemolytica pneumonias. A mutant Gram-negative bacterin significantly attenuated the clinical responses of calves challenged with E. coli; Pasteurella multocida and Pasteurella hemolytica endotoxins.

Increased calving intervals have been associated with low levels of anti-endotoxin antibody in diary cows. Although increased calving internals are a more subtle manifestation of Gram-negative endotoxemia, they do suggest that sublethal endotoxemia may cause early embryonic death and aberrant cycling in cows. Reducing the incidence of endotoxemia may, therefore help eliminate these problems.

The results of several recent studies conducted in cattle, horses, and swine strongly suggests that elevating the levels of cross-protective anti-endotoxin (anti-core) antibodies in the serum is crucial to alleviating serious clinical responses to Gram-negative endotoxemia. Apparently these antibodies can best be provided through an individual’s own immune system when it is exposed to a lipopolysaccharide antigen supplied by a mutant Gram-negative bacterin. Mutant strains of S. typhimurium and E. coli used in recent studies produced cross protective and anti-endotoxin antibodies in domestic animals and man.

The widespread use of Gram-negative cross-protective vaccines offers the possibility of significantly reducing the economic losses associated with Gram-negative septicemias and endotoxemias in food and companion animals.

Endotoxemia and endotoxic shock are serious complications of coliform mastitis. Endotoxemia results from the release of endotoxins through the death of Gram-negative bacteria, such as E. coli during phagocytosis by udder leukocytesl or by the action of antimicrobials used in treatment. The clinical signs of coliform mastitis include serous secretion in the affected quarter or quarters, pyrexia, depression, anorexia, swelling and firmness of the affected quarter or quarters, ruminal hypomotility, muscle fasciculations, cold skin temperature, and diarrhea–all signs of endotoxemia.

Traditionally, treatment of coliform mastitis has been initiated only after the development of clinical illness. Therapy has been limited to the use of anti-inflammatory agents, fluid therapy, and combinations of antimicrobials such as oxytetracycline, chloramphenicol, gentamicin, kanamycin, and polymyxin B.

The chief disadvantage of treatment initiated after clinical illness has developed is that the disease has frequently advanced to an irreversible state. Moreover, this treatment requires withholding the cow’s milk from market for days to weeks, depending on the type and amount of drug used to counter the infection. And even with successful treatment, only 20% of mastitis cows ever return to normal production; most are culled for agalactia. Also of recent concern is the development of drug-resistant salmonellae with the potential for entry into the food chain.

Two methods are currently available for decreasing the prevalence of coliform mastitis. First, better management of bedding and teat sanitation techniques decreases the exposure of teat ends to bacteria. Second, vaccination enhances the cow’s immunologic resistance to environmental bacteria.

Previously, vaccines were limited to three types: autogenous bacterial isolates expressing various specific antigens (K antigens or O-carbohydrate side chains), live vaccines composed of attenuated or deletion-modified bacteria, and polyvalent vaccines composed of the serotypes sometimes associated with mastitis .

But the use of autogenous vaccines is often neither timely nor cost-or production-efficient because such vaccines are manufactured after the disease has developed–too late for the affected herd to develop active immunity. Live vaccines may revert to the wild-type parental strain and thereby become pathogenic for vaccinated animals. And the primary disadvantage of polyvalent vaccines composed of multiple bacterial isolates expressing various antigenic epitopes (K antigens or O-carbohydrate side chains) is that the bacterial isolates causing disease are subject to epidemiologic shifts and drifts in antigenic epitopes. If a shirt or drift occurs, the vaccine is no longer efficacious.

Moreover, the K and O-carbohydrate (LPS/endotoxin) antigens are potent stimulators of inflammation. O-carbohydrate antigens are released on bacteriolysis in E. coli mastitis increasing mammary blood flow and contributing to marked swelling of the gland.11 Absorption into the blood stream can cause high fever, depression, leukopenia followed by leukocytosis, prolonged hypoglycemia and, in severe cases, irreversible shock and death of the mastitic cow.

Cross-protective vaccines have been manufactured using genetically engineered mutants such as the patented R/17 strain of Salmonella typhimurium and the J-5 strain of E. coli. ENDOVAC-Dairy® & ENDOVAC-Beef®(IMMVAC), manufactured with the R/17 mutant and combined with an immune-potentiating adjuvant, significantly reduces the devastating diseases caused by Gram-negative bacteria producing various endotoxins.

The patented R/17 mutant is an Re mutant totally devoid of O- carbohydrate side chains and is referred to as a “naked-core” mutant. Vaccines composed of Re mutant, or naked core mutants, expose the core antigens of the bacterial cell wall to the host’s immune system for the subsequent production of cross-protection antibodies. This circumvents undesirable anaphylaxis and the specific problems associated with the polyvalent vaccines. These cross-protection antibodies aid the host’s liver in neutralizing E. coli, Salmonella typhimurium, Pasteurella multocida and Pasteurella hemolytica endotoxins. What’s more, the naked-core vaccines stimulate opsonizing antibodies that enhance phagocytosis of the E. coli bacteria causing coliform mastitis.