Gram-negative endotoxemia contributes to the signs associated with coliform mastitis2, diarrhea septicemias,5 and pneumonias9 in cattle.

The signs of endotoxemia can be alleviated by anti-endotoxin antibodies.5 An enzyme-linked immunosorbent assay of sera from control and vaccinated calves showed that antibodies produced in response to a mutant Salmonella typhimurium bacterin-toxoid reduced the clinical responses to both Escherichia coli and Pasteurella endotoxins,5 thus achieving cross-protection via core antigen technology. With one vaccine you receive full protection from E.coli, Salmonella, and Pasteurella.

Lipid A, the toxic moiety of endotoxin, appears to be identical in all gram-negative bacteria, although endotoxins from different gram-negative bacteria exhibit slightly different toxicities. Differences are related to the biochemical arrangement and complexity of the sugars and side chains that compose the mucous capsule of different gram-negative bacteria. These sugars and side chains are covalently bonded to Lipid A through keto-deoxyoctanoic acid.

The current serotyping system automatically associates E. coli serotype K99 with calf diarrhea, Pasteurella haemolytica 1 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-deoxyoctanoic acid) in their cell walls. This common core offers the opportunity to genetically engineer workable cross-protective vaccines.6

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)10,13 or all (S. typhimurium R/17)17 of their capsular or “O” side-chain carbohydrates. Vaccines prepared with these rough mutants expose the bacterium’s usually protected naked core to the cow’s immune system. Because this core is common to all Enterobacteriaceae and to most other gram-negative bacteria, humoral (B-lymphocyte stem cells) and cell-mediated (T-lymphocyte stem cells) immunity elicited by these genetically engineered vaccines is cross-protective for essentially all gram-negative bacterial diseases. These vaccines are used in conjunction with some of the new immune modulators (e.g., IMMUNEPlus® and muramyl dipeptide), which selectively potentiate the B- and T-lymphocyte stem cells.17

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 withdrawal time. Often, 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.14 Vaccination, therefore, would seem to offer a better method for managing Coliform mastitis.

In dairy cows, Coliform mastitis is most commonly associated with E. coli bacteria and endotoxins.10 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.

A logical approach, then, 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 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. Independent studies of California and Arizona dairy cows, for example, have shown that mutant gram-negative bacterins lowered the incidence and severity of Coliform mastitis.10,8

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 leukocytes3,7 or by the action of antimicrobials used in treatment.11 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 hypo-motility, muscle fasciculation, cold skin temperature, and diarrhea – all signs of endotoxemia as well.

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 initiating treatment 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 several days, depending on the type and amount of drug used to counter the infection. Even with successful treatment, only 20% of mastitis cows ever return to normal production. Most are culled for agalactia.15 An additional 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 decrease 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 serotypes sometimes associated with mastitis.

Often, the use of autogenous vaccines is neither timely nor cost- or production-efficient because such vaccines are manufactured after the disease has developed. This is 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. 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 shift 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.26 Absorption into the blood stream can cause high fever, depression, and leukopenia, followed by leukocytosis, prolonged hypoglycemia, and, in severe cases, irreversible shock and death of the mastitic cow.27

Diarrhea invariably alters the balance of the intestinal microflora. Impaired movement of luminal contents and increased water content of evacuated feces decrease the numbers of lactobacilli and homofermentative streptococci. Accompanying this change is a connected increase in the 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 diarrhea syndrome, it creates a potential for life-threatening, irreversible hemorrhagic shock, disseminated intravascular coagulation, and acute oliguric renal failure.

Increased calving intervals have been associated with low levels of anti-endotoxin antibody in dairy cows.19 Although increased calving intervals 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. Therefore, reducing the incidence of endotoxemia will help eliminate these problems.

Colostrums can be enhanced with good cow vaccination. More antibody (higher titer) in the colostrums means more antibody passed on to the calf, thus creating longer duration of immunity in the calf.21