Protection of Ponies from Heterologous and Homologous Endotoxin challenges via Salmonella Typhimurium Bacterin-Toxoid


Twenty normal ponies were vaccinated twice with a Salmonella typhimurium bacterin-toxoid at 14-day intervals. Simultaneously, 20 normal control ponies each received two injections of dialuminum trioxide/saline placebo at 14-day intervals. Fourteen days following the vaccination booster or the second placebo injection, 20 ponies – 10 vaccinated and 10 treated with placebo injection – were challenged with 10 ng/kg of S. typhimurium endotoxin. Similarly, the other 20 ponies – 10 vaccinated and 10 treated with placebo – were challenged with 50 ng/kg of Escherichia coli 055:B5 endotoxin. There was a significant difference (P < 0.05) between the clinical responses of the vaccinated and placebo-treated groups challenged with either S. typhimurium or E. coli 055:B5 endotoxin as measured by the endotoxin colic index, mean anorexia time intervals, and IgG(t) antibody titers. Tables and bar graphs are included.

Protection of Ponies from Heterologous and Homologous Endotoxin challenges via Salmonella Typhimurium Bacterin-Toxoid

Ronald F. Sprouse, Ph.D.
Department of Pathology
School of Medicine
Harold E. Garner, D.V.M., Ph.D.
Kris Lager, B.S., M.S.
Department of Veterinary Medicine and Surgery
College of Veterinary Medicine
University of Missouri

Columbia, Missouri 65201


Some of the most common and most devastating diseases encountered by the equine practitioner are those associated with Gram-negative endotoxins. Colic, diarrhea caused by Gram-negative organisms, dystocia/metrtis, and carbohydrate overload are often complicated by Gram-negative endotoxins.1,2 Failure of passive transfer is the primary predisposing factor to neonatal septicemia, which is caused most frequently by Gram-negative bacteria.1 The host’s biological responses to endotoxins result in many of the recognizable clinical signs exhibited and often culminate in death.3-6 An active immunization strategy aimed at host inactivation of endotoxin represents a rational approach for preventing the devastating effects of endotoxemia.
Immunologic strategies that would provide protection from the overwhelming effects of Gram-negative endotoxemia have been difficult to develop.7 The specific serotypes of endotoxin involved in a case of endotoxemia may be one ore more members of the large Gram-negative family Enterobacteriaceae. Because there are hundreds of serotypes, it is impractical to combine sufficient autogenous vaccines to provide broad-spectrum protection. Thus, the need for a single-source bacterin that provides cross-protection against virtually all Gram-negative endotoxins is obvious.
The fact that almost all species of Gram-negative bacteria possess analogous cell wall characteristics has provided the basis for many immunological studies conducted over the past 20 years.7-13 R-mutants of Salmonella sp. and Escherichia coli have been the focus of the majority of these studies.8,10
R-mutants are “rough”-appearing cell colonies of mutant Gram-negative bacteria. These mutants are biochemically characterized by their relative absence of oligosaccharides (“O”) side chain attachments. The relative degree of “O” side chain absence is designated by the capital letter R accompanied by the small case letters “a” through “e” with Re completely lacking “O” side chains.8,10,11 The J5 E. coli mutant previously studied by us and others is characterized as Rc and thus possesses “O” side chains.
Removal of these “O” side chains via mutation allows the core antigen of the cell wall to be presented to the immune system for the subsequent production of cross-protective antibodies. Problems associated with specific serotype characteristics are thereby circumvented. Antibodies formed in response to these core antigens, which are devoid of the “O” side chains, can cross-protect an individual from many and possibly all Gram-negative endotoxins.
An Re-type mutant bacterial strain from a parent Salmonella typhimurium was isolated from a horse suffering from Salmonellosis.14 This naked core Re-mutant was combined with a toxoid and dialuminum trioxide to make a cross-protective vaccine.15 The results of homologous and heterologous efficacy testing in equids immunized with this vaccine are presented.

Materials and Methods


The vaccine (Endovac-Equi™: IMMVAC, Inc., Columbia, MO) used in these experiments contained a killed bacterial Re-mutant of S. typhimurium (bacterin), an immune modulator (endotoxoid), and a protein/lipid binding carrier/adjuvant (dialuminum trioxide). Each pony was vaccinated and boostered within 2 weeks with either the vaccine or a dialuminum trioxide/saline placebo. Each pony was challenged intravenously with endotoxin 2 weeks following the booster injection.


Forty healthy ponies ranging from 2 to 14 years in age and 79 to 200 kg (174 to 440 lb) weight were used in this study. The 17 mares, 21 geldings, and two stallions were divided as evenly as possible into two groups of 20 on the basis of sex and then randomized into four groups of 10. Each animal in two groups of 10 was administered two 1.6 ml doses of the vaccine into the cervical musculature 14 days apart. (Note: 1.6 ml represents 80% of the recommended 2.0 ml dose, required by the USDA for official efficacy testing of the vaccine.) Animals in the other two groups of 10 were each administered two 1.6 ml doses of a 50% dialuminum trioxide/50% saline placebo intramuscularly 14 days apart. This experimental design allowed each group of 10 animals that received the vaccine to be compared to a group of 10 animals that received placebo when all were challenged with endotoxin.
Twenty ponies, 10 vaccinated with the vaccine and 10 injected with the placebo, were challenged with an intravenous bolus of 10 ng/kg of S. typhimurium endotoxin. The other 20 ponies, 10 vaccinated with the vaccine and 10 injected with the placebo, were challenged with an intravenous bolus of 50 ng/kg of E. coli 055:B5 endotoxin. Each pony was fasted for 12 hours prior to endotoxin challenges but the animals were allowed free access to water until placed in a cross tie for observation. Each pony was observed 20 minutes prior to endotoxin injection to establish control behavior and 1 hour following injection to observe clinical responses. Responses were recorded continuously. In addition, during the second hour following endotoxin administration, each pony was turned loose in a box stall with free access to alfalfa hay and observed to determine whether or not it was anorexic.


The endotoxin colic index scoring method used to generate the data in Figures 1 and 2 was established prior to the present study by statistically analyzing the observations of three people separately recording the clinical signs exhibited by 56 head of cross-tied horses and ponies 20 minutes prior to and 1 hour following intravenous bolus administration of varying dosage levels of either Salmonella sp. or E. coli endotoxin. Pawing, kicking, leg flexing, stretching, bowing stretches, looking at the flank, pinning the ears back and tail switching along with CNS depression progressing to comatosis were all included as signs used to describe the progression of behavior which ranged from level 1.0 to 8.0 of the endotoxin colic index. During efficacy studies, the assessment of the observations was accomplished via a blinded scorer. All of the horses, whether they possessed protective levels of anti-core-antigen antibodies or not, exhibited signs that approached level 3.0 when they were scored. The unprotected animals developed sufficient clinical signs to progress through level 3.0 and higher while those that were protected exhibited colic index scores of 3.0 or lower.


Serum samples collected from each pony prior to and 4 weeks following the first injection of vaccine or placebo were analyzed by an ELISA assay adapted from a previously developed radioimmunoassay (RIA) for specific IgG(t) anti-endotoxin antibody levels used to establish the endotoxin colic index.16,17 The technician who analyzed the pre- and post-vaccination serum samples for anti-core-antigen antibody levels was not aware of any animal’s grouping.


Data were analyzed via analysis of variance statistical techniques. The predetermined acceptable level of probability was 0.5 or less.


When challenged with endotoxin, ponies vaccinated with the S. typhimurium bacterin-toxoid compared to those injected with the placebo were significantly (P < 0.05) different in terms of the mean endotoxin colic index scores reflecting colicky pain and somnolence, anorexia time intervals and IgG(t) antibody levels (Table 1 & 2 and Figs. 1-4). In Tables 1 & 2, the line “3.0” represents the previously established threshold that divided the individuals that possessed protective levels of anticore antigen antibodies from those individuals that did not.
The mean endotoxin colic index scores of immunized versus placebo-injected groups homologously challenged with S. typhimurium endotoxin were significantly (P < 0.001) different (Table 1, Fig. 1). The difference between these groups in terms of either mean anorexia time intervals or mean IgG(t) antibody titers (Table 1, Fig. 3) was also significant (P < 0.05 and P < 0.001 respectively).
Similarly, the mean endotoxin colic index scores of immunized versus placebo-injected groups heterologously challenged with E. coli 055:B5 endotoxin were significantly (P < 0.001) different (Table 2, Fig. 2). The differences between these groups in terms of either mean anorexia time intervals (P < 0.05) or mean IgG(t) antibody titers (P < 0.001) were significant, also (Table 2, Fig. 4).
In this study, 90% of the ponies that received the vaccine exhibited a transient, localized swelling 1 to 10 cm in diameter at the cervical musculature injection site 2 to 4 days post injection. Four of the 20 vaccinates exhibited suppressed appetites for 2 to 3 days post vaccination. None required treatment.


The increase in serum IgG(t) antibody levels in the vaccinated pony groups apparently provided the active immunity responsible for protection against the outward clinical effects of the endotoxin challenges. It is interesting that these results confirmed the results from other laboratories when various species were vaccinated with similar Gram-negative mutant bacterins and challenged with heterologous endotoxins.9-11,18 It is also important to note that the protection provided by the antibodies produced in response to the core antigen of the Re-mutant S. typhimurium bacterin-toxoid cross-protected the ponies from the heterologous E. coli 055:B5 endotoxin as well as from the homologous S. typhimurium endotoxin.
The dialuminum trioxide adjuvant in this vaccine stimulated the localization of macrophages in the muscular tissue at the injection site. The macrophage-processed antigen then slowly leaked out of the localized macrophages, providing a prolonged antigenic stimulus.19 Therefore, a 1 to 10 cm local reaction was expected following injection of the vaccine and was indicative of a viable host immunization. Dialuminum trioxide influenced the primary immune response and helped maintain the other two vaccine components, i.e., bacterin and toxoid in suspension.
The toxoid portion of the combination cross-protective vaccine stimulates the B-lymphocytes to divide and produce antibodies directed against the naked core determinant while the killed Re-mutant bacterial cells (bacterin) provided the naked core determinant to serve as antigen for antibody production.
Since these efficacy studies, field study observations have confirmed that injection of the vaccine resulted in lesser local responses when injected into the musculature of the rear quarters14 and that moderate exercise of the vaccinates was beneficial. Any rise in body temperature and/or generalized muscular soreness was treated with non-steroid anti-inflammatories and other appropriate supportive measures. In accordance with USDA recommendations, any horse that suffers an allergic response following vaccination should be treated with epinephrine or its equivalent. No allergic responses were discerned during the efficacy and subsequent field studies.
Equids with obvious chronic laminitis or those that have suffered an endotoxic crisis within the last 60 days do not significantly respond immunogenically to vaccination with the cross-protective vaccine and thus should not be vaccinated with it.14

In conclusion, the active immunization of healthy horses with a cross-protective vaccine can aid the control of Gram-negative endotoxemia. A transient, localized response at the site of intramuscular injection will most likely occur in the majority of vaccinates. Therefore, it is imperative that veterinarians administering the vaccine inform owners and/or trainers about the expected local response and the importance of providing moderate exercise daily for at least 10 days following vaccination.

The authors extend their gratitude to Dorothy Brandon, Dan Hatfield, Joe Miramonti, Anne Sears, Kelly Lager, Bill Starke, Patsy McClenahan, and Carol Skinner for their expert technical assistance.
Funded in part by the University of Missouri College of Veterinary Medicine, School of Medicine, and IMMVAC, Inc., Columbia, Missouri.

1. Carter GK, Martens RJ: Septicemia in the Neonatal Foal. Comp Cont Ed Pract Vet 8:5256-5271, 1986.
2. Sprouse RF, Garner HE, et al : Plasma Endotoxin Levels in Horses Subjected to Carbohydrate Induced Laminitis. Eq Vet J 19:25-28, 1987.
3. McCarty DO, Kluger MJ, et al: The Role of Fever in Appetite Suppression After Endotoxic Administration. Am J Clin Nutr 40:310-316, 1984.
4. Moldawer LL, Georgiett M, et al: Interleukin 1, Tumor Necrosis Factor-alfa (Cachetin) and the Pathogenesis of Cancer Cachexia. Clin Phys 7:263-274, 1987.
5. Movat HZ, Cybulsky MI, et al: Acute Inflammation in Gram-Negative Infection: Endotoxins, Interleukin 1, Tumor Necrosis Factor and Neutrophils. Fed Proc 46:97-104, 1987.
6. Hart BL: Animal Behavior and the Fever Response: Theoretical Considerations. J Am Vet Med Assoc 187:998-1001, 1985.
7. Morris DD, Whitlock RH, et al: Endotoxemia in Horses: Protection Provided by Antiserum to Core Lipopolysaccharide. Am J Vet Res 47:544-550, 1986.
8. McCabe WR, Kreger M, Johns MA: Type-Specific and Cross-Reaction Antibodies in Gram-Negative Bacteremia. New Engl J Med 287:262, 1972.
9. Ng AK, Chan LH, et al: Relationship of Structure to Function in Bacterial Endotoxins: Serologically Cross-Reactive Components and Their Effect on Protection of Mice Against Some Gram-Negative Infections. J Gen Mic 94:107-116, 1976.
10. Braude AI: Endotoxic Immunity. Adv Intern Med 26:427-445, 1980.
11. Cullor JS, Fenwick BW, et al: Protection from Endotoxic Shock in Calves by Antibodies Against Common LPS Core Antigens Induced by Immunization with E. coli (J5). Conf Res Work (An Dis abstr) #48, 1984.
12. Marget W, Mar PJ, et al: Preliminary Study on Administration of High-Titer Lipid A Antibody Serum in Sepsis and Septic Shock Patients. Infection 13:120-124, 1985.
13. Young LS, Stevens P, et al: Functional Role of Antibody Against ‘Core’ Glycolipid of Enterobacteriaceae. J Clin Invest 56:850-861, 1975.
14. Sprouse RF, Garner HE, et al: Active Immunity, Passive Immunity and Passive Transfer: Gram Negative Endotoxemia Cross Protection Studies Using Salmonella typhimurium Bacterin-Toxoid and Antiserum in Horses. Univ of Missouri. Unpublished data, 1988.
15. Garner HE, Sprouse RF, et al: Active and Passive Immunization for Blockade of Endotoxemia. Am Assoc Eq Pract 31:525-532, 1985.
16. Garner HE, Sprouse RF, et al: Cross Protection of Ponies from Sublethal Escherichia coli Endotoxemia by Salmonella typhimurium Antiserum. Eq Prac 10(4):10-17, 1988.
17. Reardon TP, Sprouse RF, et al: Radioimmunoassay for the Detection of Antigen-Specific IgM, IgG, and IgA in Equine Sera. Am J Vet Res 43:294-298, 1982.
18. Cullor JS, Spier SJ, et al: Antibodies That Recognize Gram-Negative Core Antigens: How Important Are They? Proc of ACVIM 1988 pp 503-508.
19. Tizard I: Veterinary Immunology, An Introduction, 3rd Edition. Philadelphia, WB Saunders Company, 1987.