Category: Dairy Case Studies

Studies:  E. coli and P. multocida Endotoxin Challenge Study

AGRI-PRACTICE – COW/CALF – DISEASE CONTROL

 

At 14-day intervals, 12 calves were vaccinated twice with a S. typhimurium bacterin-toxoid, and 12 control calves each received two injections of dialuminum trioxide/saline placebo. Two weeks following the vaccination booster, or the second placebo injection, ten calves – five vaccinated and five treated with placebo – were challenged with 100 ng/kg of E. coli 055:B5 endotoxin. Similarly, another 14 calves – seven vaccinated and seven placebo-treated – were challenged with 50 ng/kg of P. multocida endotoxin. There was a significant difference (P < 0.05) between the clinical responses of the vaccinated and placebo-treated group challenged with either E. coli 055:B5 or P. multocida endotoxin as measured by the endotoxin colic index, mean anorexia time intervals, and IgG(t) serum antibody titers.

 

Cross-Protection of Calves from

AGRI-PRACTICE – COW/CALF – DISEASE CONTROL

 

At 14-day intervals, 12 calves were vaccinated twice with a S. typhimurium bacterin-toxoid, and 12 control calves each received two injections of dialuminum trioxide/saline placebo. Two weeks following the vaccination booster, or the second placebo injection, ten calves – five vaccinated and five treated with placebo – were challenged with 100 ng/kg of E. coli 055:B5 endotoxin. Similarly, another 14 calves – seven vaccinated and seven placebo-treated – were challenged with 50 ng/kg of P. multocida endotoxin. There was a significant difference (P < 0.05) between the clinical responses of the vaccinated and placebo-treated group challenged with either E. coli 055:B5 or P. multocida endotoxin as measured by the endotoxin colic index, mean anorexia time intervals, and IgG(t) serum antibody titers.

 

Cross-Protection of Calves from E. coli and P. multocida Endotoxin Challenges Via S. typhimurium Mutant Bacterin-Toxoid*

 


Ronald F. Sprouse, Ph.D.

Department of Pathology

School of Medicine

University of Missouri

Columbia, Missouri 65201

 

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

 

Introduction

 

Some of the most common and devastating diseases encountered by the bovine practitioner are those associated with endotoxins. Gram-negative diarrheas and pneumonias are often complicated by endotoxins.1,2 Failure of passive transfer of uniformity 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 gram-negative endotoxins represents a rational approach for preventing the devastating effects of endotoxemia.

Immune strategies that would aid cattle by providing cross-protection from the overwhelming effects of various gram-negative endotoxemias have been difficult to develop.7 In a case of endotoxemia, the specific serotype sources of endotoxin involved may be from one or more members of the large gram-negative family, Enterobacteriaceae. Because there are hundreds of gram-negative serotypes, it would be impractical to combine sufficient autogenous vaccines to provide broad-spectrum protection. Thus, a single source bacterin that provides cross-protection against virtually all gram-negative endotoxins is needed.

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.7-15 R-mutants of Salmonella sp. and Escherichia coli have been the focus of many 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 lowercase letters “a” through “e” with Re completely lacing “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 allowed the core antigen of the cell wall to be presented to the immune system for the subsequent production of cross-protective antibodies,15,16 thereby, circumventing problems associated with specific serotype characteristics. Antibodies formed in response to these core antigens devoid of the “O” side chains can cross-protect an animal from many, and possibly all, gram-negative endotoxins.

An Re-type mutant bacterial strain from a parent Salmonella typhimurium was engineered to form an Re-type mutant that possesses no “O” side chains. This naked core Re-mutant was combined with a toxoid and dialuminum trioxide to make a cross-protective vaccine.16 The results of Heterologous efficacy testing in calves immunized with this vaccine are presented.

 

Materials and Methods

 

 

The vaccine used in these experiments contained a killed bacterial Re mutant of S. typhimurium (bacterin), and immune modulator (endotoxin), a protein/lipid binding carrier/adjuvant (dialuminum trioxide), and oil. Each calf was vaccinated and boostered within 2 weeks either with the vaccine or a dialuminum trioxide/saline placebo. Each calf was intravenously challenged with endotoxin 2 weeks post-booster injection.

Twenty-four healthy calves ranging from 3 to 4 months in age and 79 kg to 200 kg in body weight were used in this study. The 22 bulls and two heifers were divided as evenly as possible into two groups of 14 and 10, respectively, on the basis of sex and then randomized into two groups of seven and two groups of five. One group of seven and one group of five were administered two 1.6 ml doses of the vaccine into the cervical musculature 14 days apart. The United States Department of Agriculture required 80%, or 1.6 ml doses of a 50% dialuminum trioxide/50% saline placebo intramuscularly 14 days apart. This experimental design allowed each group that received the vaccine to be compared with a group that received placebo when all were challenged with endotoxin.

Ten calves, five vaccinated with the bacterin-toxoid and five injected with the placebo, were challenged with an intravenous bolus of 100 ng/kg of E. coli 055:B5 endotoxin. The other 14 calves, seven vaccinated with the bacterin-toxoid and seven injected with the placebo, were challenged with an intravenous bolus of 50 ng/kg of Pasteurella multocida endotoxin. Each calf was fasted for 12 hours prior to endotoxin challenges but was allowed free access to water until tied in a box stall for observation. Each calf was observed 60 minutes prior to endotoxin injection to establish control behavior and was then allowed free choice of alfalfa and observed for 1 hour following endotoxin injection to observe clinical responses. Responses were continuously recorded. In addition, during the second hour following endotoxin administration, each calf was turned loose in a box stall and allowed free choice of alfalfa, hay, and water and closely 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 individuals recording the clinical signs exhibited by 30 head of tied calves for 1 hour prior to and 1 hour following intravenous bolus administration of varying dosage levels of either Pasteurella or E. coli endotoxin.12 Kicking, leg flexing, stretching, bowing-stretches, looking at flank, hyperpnea, and dyspnea 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 Level 6.0 of the endotoxic colic index. During efficacy studies, the assessment of the observations was accomplished via a blinded scorer. All of the calves, whether they possessed protective levels of anticore-antigen antibodies or not exhibited signs that approached Level 2.0 when they were scored. The unprotected animals developed sufficient clinical signs to progress through level 2.0 and higher, while those that were protected exhibited colic index score levels of less than 2.0.

Serum samples collected from each calf before and 4 weeks following the first injection of vaccine or placebo were analyzed for the present study by an ELISA assay adapted from a previously developed radioimmunoassay (RIA) for specific IgG(t) antiendotoxin antibody levels.17,18 The technician that analyzed the pre- and post-vaccination serum samples for anticore-antigen antibody levels was not aware of any animal’s category.

Data were analyzed via analysis of variance statistical techniques. The predetermined acceptable probability level was 0.05 or less.

 

Results

 

When challenged with endotoxin, calves vaccinated with the S. typhimurium bacterin-toxoid compared with those injected with the placebo were significantly (P < 0.05) different in terms of the mean endotoxin colic index scores reflecting colicky pain, dyspnea and somnolence, mean IgG(t) antibody levels, and anorexia time intervals (Tables 1 & 2; Figs. 1, 2, 3, & 4). The line 2.0 represents the previously established threshold that divided those with protective levels of anticore-antigen antibodies from those without.12 The mean endotoxin colic respiratory index scores of immunized vs. placebo-injected groups heterologously challenged with E. coli 055:B5 endotoxin were significantly (P < 0.001) different (Table 1; Figs. 1 & 2).

The differences between these groups (Table 2; Fig. 4) in terms of either mean IgG(t) antibody titers (P < 0.001) or mean anorexia time intervals (P < 0.05) were significant. Similarly, the mean endotoxin colic index scores of immunized vs. placebo-injected groups heterologously challenged with P. multocida endotoxin were significantly (P < 0.001) different (Table 1; Fig. 1). The differences between these groups in terms of either mean IgG(t) antibody tiers (P < 0.05) (Table 2; Figs. 3 & 4) or mean anorexia time intervals (P < 0.05) (Table 3) were also significant. In this study, 90% of the calves that received the vaccine exhibited a transient palpable 1-cm diameter swelling in the cervical musculature injection site 2 to 4 days postinjection, which was nonpalpable 2 weeks following injection. None required treatment or went off feed.

 

Discussion

 

The increase in serum IgG(t) antibody levels in the vaccinated calf groups apparently provided the active immunity responsible for protection against the outward clinical effects of the heterologous endotoxin challenges. It is interesting that these results confirmed the results of other laboratories when various species were vaccinated with similar gram-negative mutant bacterins and challenged with Heterologous endotoxins.9-11,19 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 calves from the heterologous E. coli 055:B5 endotoxin challenge as well as from the heterologous P. multocida endotoxin challenge.

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.20 Therefore, a local response was expected following injection of the vaccine and was indicative of a viable hose immunization. Dialuminum trioxide influenced the primary immune response and helped maintain the other two vaccine components in suspension.

The toxoid portion of the combination cross-protective vaccine stimulate 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 conducting these efficacy studies, results of field study observations, including a rise in body temperature and/or generalized muscular soreness, were not detected in any calves or cows. In accordance with USDA recommendations, any animal that suffers an allergic response following vaccination should be treated immediately with epinephrine or its equivalent. No allergic responses were discerned during the efficacy and subsequent field studies.

 

Conclusion

 

The anticore-antigen antibody efficacy demonstrated in this study offers new possibilities for aiding in the control of end-stage consequences of such gram-negative diseases as E. coli sp. diarrhea, Salmonella sp. diarrhea, and Pasteurella sp. pneumonia. Because of the cross-protectiveness of the antibodies demonstrated in this study, it is suspected that cattle can also be protected from endotoxins arising from other gram-negative bacteria such as Klebsiella sp., Enterobacterieae sp. Proteus sp., and others. Application of this technology may add a new dimension to immunologic control of economically important gram-negative bovine diseases.

 

ACKNOWLEDGEMENTS

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.

This study was funded in part by the University of Missouri College of Veterinary Medicine, School of Medicine, and IMMVAC, Inc., Columbia, Missouri.

 

REFERENCES

  1. Carter GK, Martens RJ: Septicemia in the Neonatal Foal. Comp Cont Ed Pract Vet 8:5256-5271, 1986.
  2. Sprouse RF, Garner HE, Green EM: 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, Lanholm K: Interleukin 1, Tumor Necrosis Factor-alfa (Cachetin) and the Pathogenesis of Cancer Cachexia. Clin Phys 7:263-274, 1987.
  5. Movat HZ, Cybulsky MI, Golditz IG, 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, Cullor JS, Whitlock RH: 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, Chang CM, 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, Williams MR, 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. Lager KL: Development and Application of Behavioral Indices for Evaluation of Equine and Bovine Responses to Low Level Endotoxin Challenges. Master’s Thesis, University of Missouri, 1989.
  13. Marget W, Mar PJ, Jaspers L, et al: Preliminary Study on Administration of High-Titer Lipid A Antibody Serum in Sepsis and Septic Shock Patients. Infection 13:120-124, 1985.
  14. Young LS, Stevens P, Ingram J: Functional Role of Antibody Against ‘Core’ Glycolipid of Enterobacteriaceae. J Clin Invest 56:850-861, 1975.
  15. Sprouse RF, Garner HE, Lager KS: Protection of Ponies from Heterologous and Homologous Endotoxin Challenges Via Salmonella Typhimurium Bacterin Toxoid. Eq Pract 11:34-40, 1988.
  16. Garner HE, Sprouse RF, Green EM: Active and Passive Immunization for Blockade of Endotoxemia. Am Assoc Eq Pract 31:525-532, 1985.
  17. Garner HE, Sprouse RF, Lager KS: Cross Protection of Ponies from Sublethal Escherichia Coli Endotoxemia by Salmonella Typhimurium Antiserum. Eq Pract 10(4):10-17, 1988.
  18. Reardon TP, Sprouse RF, Garner HE: Radioimmunoassay for the Detection of Antigen-Specific IgM, IgG, and IgA in Equine Sera. Am J Vet Res 43:294-298, 1982.
  19. Cullor JS, Spier SJ, Tyler JW, Smith BP: Antibodies that Recognize Gram-Negative Core Antigens: How Important Are They. Proc of ACVIM 1988, pp 503-508.
  20. Tizard, Ian: Veterinary Immunology: An Introduction. 3rd Ed. Philadelphia, WB Saunders Co., 1987.

 

TABLE 1
Comparison of Mean Endotoxin Respiratory Colic Index Scores and Serum IgG Antibody Titers of E. coli

Endotoxin-Challenged, Placebo-Treated, and Vaccinated Calves

 
Endotoxin-Challenged Calves
Parameter Placebo (control); N = 5 Vaccinate; N = 5
Mean Endotoxin Colic Respiratory Index Scorea
Mean 2.31 0.32c
SD ± 2.40 ± 0.64
SEM ± 1.20 ± 0.30
Range 0.60-3.6 0.0-1.0
Mean Serum IgG Titer (Log 2)b        
  Pre- Post- Pre- Post
Mean 8.60 9.00d 9.80 12.20c
SD ± 1.20 ± 1.55 ± 1.47 ± 1.17
SEM ± 0.60 ± 0.77 ± 0.74 ± 0.59
Range 8-11 8-12 8-11 11-14

a Endotoxin respiratory colic index scores were analyzed via three-factor analysis of variance with repeated measures on one factor.

b Serum IgG antibody measurements were analyzed via two-factor analysis of variance techniques with repeated measures on one factor.

c Mean value significantly (p < 0.05) different from control or pretreatment values.

d Mean value not significantly (p > 0.05) different from pretreatment values.

SE = Standard deviation; SEM = Standard error of the mean.

 

TABLE 2
Comparison of Mean Endotoxin Respiratory Colic Index Scores and Serum IgG Antibody Titers of Pasteurella

Endotoxin-Challenged, Placebo-Treated, and Vaccinated Calves

 
Endotoxin-Challenged Calves
Parameter Placebo (control); N = 7 Vaccinate; N = 7
Mean Endotoxin Colic Respiratory Index Scorea
Mean 2.35 0.64c
SD ± 2.26 ± 1.17
SEM ± 0.92 ± 0.48
Range 1.0-3.7 0.10-2.1
Mean Serum IgG Titer (Log2)b        
  Pre- Post- Pre- Post
Mean 9.29 9.71d 8.29 13.10c
SD ± 1.28 ± 1.03 ± 0.45 ± 1.36
SEM ± 0.52 ± 0.42 ± 0.18 ± 0.56
Range 8-11 8-11 8-9 11-15

a Endotoxin respiratory colic index scores were analyzed via three-factor analysis of variance with repeated measures on one factor.

b Serum IgG antibody measurements were analyzed via two-factor analysis of variance techniques with repeated measures on one factor.

c Mean value significantly (p < 0.05) different from control or pretreatment values.

d Mean value not significantly (p > 0.05) different from pretreatment values.

SE = Standard deviation; SEM = Standard error of the mean.

 

 

 

TABLE 3
Comparison of Combined Anorexia Time Intervals of E. coli and

Pasteurella Endotoxin-Challenged, Placebo-Treated, and Vaccinated Calves

 
Endotoxin-Challenged Calves
Parameter Placebo (control); N = 12 Vaccinate; N = 12
Mean Anorexia Time Interval (minutes)a
Mean 98.1 63.0b
SD ± 22.2 28.8
SEM ± 6.4 ± 8.31
Range 48-112 29-102

a Anorexia time interval measurements were analyzed via two-factor analysis of variance techniques with repeated measures on one factor.

b Mean value significantly (p < 0.05) different from control values.

SE = Standard deviation; SEM = Standard error of the mean.s Via S. typhimurium Mutant Bacterin-Toxoid*

 


Ronald F. Sprouse, Ph.D.

Department of Pathology

School of Medicine

University of Missouri

Columbia, Missouri 65201

 

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

 

Introduction

 

Some of the most common and devastating diseases encountered by the bovine practitioner are those associated with endotoxins. Gram-negative diarrheas and pneumonias are often complicated by endotoxins.1,2 Failure of passive transfer of uniformity 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 gram-negative endotoxins represents a rational approach for preventing the devastating effects of endotoxemia.

Immune strategies that would aid cattle by providing cross-protection from the overwhelming effects of various gram-negative endotoxemias have been difficult to develop.7 In a case of endotoxemia, the specific serotype sources of endotoxin involved may be from one or more members of the large gram-negative family, Enterobacteriaceae. Because there are hundreds of gram-negative serotypes, it would be impractical to combine sufficient autogenous vaccines to provide broad-spectrum protection. Thus, a single source bacterin that provides cross-protection against virtually all gram-negative endotoxins is needed.

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.7-15 R-mutants of Salmonella sp. and Escherichia coli have been the focus of many 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 lowercase letters “a” through “e” with Re completely lacing “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 allowed the core antigen of the cell wall to be presented to the immune system for the subsequent production of cross-protective antibodies,15,16 thereby, circumventing problems associated with specific serotype characteristics. Antibodies formed in response to these core antigens devoid of the “O” side chains can cross-protect an animal from many, and possibly all, gram-negative endotoxins.

An Re-type mutant bacterial strain from a parent Salmonella typhimurium was engineered to form an Re-type mutant that possesses no “O” side chains. This naked core Re-mutant was combined with a toxoid and dialuminum trioxide to make a cross-protective vaccine.16 The results of Heterologous efficacy testing in calves immunized with this vaccine are presented.

 

Materials and Methods

 

 

The vaccine used in these experiments contained a killed bacterial Re mutant of S. typhimurium (bacterin), and immune modulator (endotoxin), a protein/lipid binding carrier/adjuvant (dialuminum trioxide), and oil. Each calf was vaccinated and boostered within 2 weeks either with the vaccine or a dialuminum trioxide/saline placebo. Each calf was intravenously challenged with endotoxin 2 weeks post-booster injection.

Twenty-four healthy calves ranging from 3 to 4 months in age and 79 kg to 200 kg in body weight were used in this study. The 22 bulls and two heifers were divided as evenly as possible into two groups of 14 and 10, respectively, on the basis of sex and then randomized into two groups of seven and two groups of five. One group of seven and one group of five were administered two 1.6 ml doses of the vaccine into the cervical musculature 14 days apart. The United States Department of Agriculture required 80%, or 1.6 ml doses of a 50% dialuminum trioxide/50% saline placebo intramuscularly 14 days apart. This experimental design allowed each group that received the vaccine to be compared with a group that received placebo when all were challenged with endotoxin.

Ten calves, five vaccinated with the bacterin-toxoid and five injected with the placebo, were challenged with an intravenous bolus of 100 ng/kg of E. coli 055:B5 endotoxin. The other 14 calves, seven vaccinated with the bacterin-toxoid and seven injected with the placebo, were challenged with an intravenous bolus of 50 ng/kg of Pasteurella multocida endotoxin. Each calf was fasted for 12 hours prior to endotoxin challenges but was allowed free access to water until tied in a box stall for observation. Each calf was observed 60 minutes prior to endotoxin injection to establish control behavior and was then allowed free choice of alfalfa and observed for 1 hour following endotoxin injection to observe clinical responses. Responses were continuously recorded. In addition, during the second hour following endotoxin administration, each calf was turned loose in a box stall and allowed free choice of alfalfa, hay, and water and closely 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 individuals recording the clinical signs exhibited by 30 head of tied calves for 1 hour prior to and 1 hour following intravenous bolus administration of varying dosage levels of either Pasteurella or E. coli endotoxin.12 Kicking, leg flexing, stretching, bowing-stretches, looking at flank, hyperpnea, and dyspnea 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 Level 6.0 of the endotoxic colic index. During efficacy studies, the assessment of the observations was accomplished via a blinded scorer. All of the calves, whether they possessed protective levels of anticore-antigen antibodies or not exhibited signs that approached Level 2.0 when they were scored. The unprotected animals developed sufficient clinical signs to progress through level 2.0 and higher, while those that were protected exhibited colic index score levels of less than 2.0.

Serum samples collected from each calf before and 4 weeks following the first injection of vaccine or placebo were analyzed for the present study by an ELISA assay adapted from a previously developed radioimmunoassay (RIA) for specific IgG(t) antiendotoxin antibody levels.17,18 The technician that analyzed the pre- and post-vaccination serum samples for anticore-antigen antibody levels was not aware of any animal’s category.

Data were analyzed via analysis of variance statistical techniques. The predetermined acceptable probability level was 0.05 or less.

 

Results

 

When challenged with endotoxin, calves vaccinated with the S. typhimurium bacterin-toxoid compared with those injected with the placebo were significantly (P < 0.05) different in terms of the mean endotoxin colic index scores reflecting colicky pain, dyspnea and somnolence, mean IgG(t) antibody levels, and anorexia time intervals (Tables 1 & 2; Figs. 1, 2, 3, & 4). The line 2.0 represents the previously established threshold that divided those with protective levels of anticore-antigen antibodies from those without.12 The mean endotoxin colic respiratory index scores of immunized vs. placebo-injected groups heterologously challenged with E. coli 055:B5 endotoxin were significantly (P < 0.001) different (Table 1; Figs. 1 & 2).

The differences between these groups (Table 2; Fig. 4) in terms of either mean IgG(t) antibody titers (P < 0.001) or mean anorexia time intervals (P < 0.05) were significant. Similarly, the mean endotoxin colic index scores of immunized vs. placebo-injected groups heterologously challenged with P. multocida endotoxin were significantly (P < 0.001) different (Table 1; Fig. 1). The differences between these groups in terms of either mean IgG(t) antibody tiers (P < 0.05) (Table 2; Figs. 3 & 4) or mean anorexia time intervals (P < 0.05) (Table 3) were also significant. In this study, 90% of the calves that received the vaccine exhibited a transient palpable 1-cm diameter swelling in the cervical musculature injection site 2 to 4 days postinjection, which was nonpalpable 2 weeks following injection. None required treatment or went off feed.

 

Discussion

 

The increase in serum IgG(t) antibody levels in the vaccinated calf groups apparently provided the active immunity responsible for protection against the outward clinical effects of the heterologous endotoxin challenges. It is interesting that these results confirmed the results of other laboratories when various species were vaccinated with similar gram-negative mutant bacterins and challenged with Heterologous endotoxins.9-11,19 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 calves from the heterologous E. coli 055:B5 endotoxin challenge as well as from the heterologous P. multocida endotoxin challenge.

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.20 Therefore, a local response was expected following injection of the vaccine and was indicative of a viable hose immunization. Dialuminum trioxide influenced the primary immune response and helped maintain the other two vaccine components in suspension.

The toxoid portion of the combination cross-protective vaccine stimulate 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 conducting these efficacy studies, results of field study observations, including a rise in body temperature and/or generalized muscular soreness, were not detected in any calves or cows. In accordance with USDA recommendations, any animal that suffers an allergic response following vaccination should be treated immediately with epinephrine or its equivalent. No allergic responses were discerned during the efficacy and subsequent field studies.

 

Conclusion

 

The anticore-antigen antibody efficacy demonstrated in this study offers new possibilities for aiding in the control of end-stage consequences of such gram-negative diseases as E. coli sp. diarrhea, Salmonella sp. diarrhea, and Pasteurella sp. pneumonia. Because of the cross-protectiveness of the antibodies demonstrated in this study, it is suspected that cattle can also be protected from endotoxins arising from other gram-negative bacteria such as Klebsiella sp., Enterobacterieae sp. Proteus sp., and others. Application of this technology may add a new dimension to immunologic control of economically important gram-negative bovine diseases.

 

ACKNOWLEDGEMENTS

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.

This study was funded in part by the University of Missouri College of Veterinary Medicine, School of Medicine, and IMMVAC, Inc., Columbia, Missouri.

 

REFERENCES

  1. Carter GK, Martens RJ: Septicemia in the Neonatal Foal. Comp Cont Ed Pract Vet 8:5256-5271, 1986.
  2. Sprouse RF, Garner HE, Green EM: 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, Lanholm K: Interleukin 1, Tumor Necrosis Factor-alfa (Cachetin) and the Pathogenesis of Cancer Cachexia. Clin Phys 7:263-274, 1987.
  5. Movat HZ, Cybulsky MI, Golditz IG, 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, Cullor JS, Whitlock RH: 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, Chang CM, 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, Williams MR, 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. Lager KL: Development and Application of Behavioral Indices for Evaluation of Equine and Bovine Responses to Low Level Endotoxin Challenges. Master’s Thesis, University of Missouri, 1989.
  13. Marget W, Mar PJ, Jaspers L, et al: Preliminary Study on Administration of High-Titer Lipid A Antibody Serum in Sepsis and Septic Shock Patients. Infection 13:120-124, 1985.
  14. Young LS, Stevens P, Ingram J: Functional Role of Antibody Against ‘Core’ Glycolipid of Enterobacteriaceae. J Clin Invest 56:850-861, 1975.
  15. Sprouse RF, Garner HE, Lager KS: Protection of Ponies from Heterologous and Homologous Endotoxin Challenges Via Salmonella Typhimurium Bacterin Toxoid. Eq Pract 11:34-40, 1988.
  16. Garner HE, Sprouse RF, Green EM: Active and Passive Immunization for Blockade of Endotoxemia. Am Assoc Eq Pract 31:525-532, 1985.
  17. Garner HE, Sprouse RF, Lager KS: Cross Protection of Ponies from Sublethal Escherichia Coli Endotoxemia by Salmonella Typhimurium Antiserum. Eq Pract 10(4):10-17, 1988.
  18. Reardon TP, Sprouse RF, Garner HE: Radioimmunoassay for the Detection of Antigen-Specific IgM, IgG, and IgA in Equine Sera. Am J Vet Res 43:294-298, 1982.
  19. Cullor JS, Spier SJ, Tyler JW, Smith BP: Antibodies that Recognize Gram-Negative Core Antigens: How Important Are They. Proc of ACVIM 1988, pp 503-508.
  20. Tizard, Ian: Veterinary Immunology: An Introduction. 3rd Ed. Philadelphia, WB Saunders Co., 1987.

 

TABLE 1
Comparison of Mean Endotoxin Respiratory Colic Index Scores and Serum IgG Antibody Titers of E. coli

Endotoxin-Challenged, Placebo-Treated, and Vaccinated Calves

 
Endotoxin-Challenged Calves
Parameter Placebo (control); N = 5 Vaccinate; N = 5
Mean Endotoxin Colic Respiratory Index Scorea
Mean 2.31 0.32c
SD ± 2.40 ± 0.64
SEM ± 1.20 ± 0.30
Range 0.60-3.6 0.0-1.0
Mean Serum IgG Titer (Log 2)b        
  Pre- Post- Pre- Post
Mean 8.60 9.00d 9.80 12.20c
SD ± 1.20 ± 1.55 ± 1.47 ± 1.17
SEM ± 0.60 ± 0.77 ± 0.74 ± 0.59
Range 8-11 8-12 8-11 11-14

a Endotoxin respiratory colic index scores were analyzed via three-factor analysis of variance with repeated measures on one factor.

b Serum IgG antibody measurements were analyzed via two-factor analysis of variance techniques with repeated measures on one factor.

c Mean value significantly (p < 0.05) different from control or pretreatment values.

d Mean value not significantly (p > 0.05) different from pretreatment values.

SE = Standard deviation; SEM = Standard error of the mean.

 

TABLE 2
Comparison of Mean Endotoxin Respiratory Colic Index Scores and Serum IgG Antibody Titers of Pasteurella

Endotoxin-Challenged, Placebo-Treated, and Vaccinated Calves

 
Endotoxin-Challenged Calves
Parameter Placebo (control); N = 7 Vaccinate; N = 7
Mean Endotoxin Colic Respiratory Index Scorea
Mean 2.35 0.64c
SD ± 2.26 ± 1.17
SEM ± 0.92 ± 0.48
Range 1.0-3.7 0.10-2.1
Mean Serum IgG Titer (Log2)b        
  Pre- Post- Pre- Post
Mean 9.29 9.71d 8.29 13.10c
SD ± 1.28 ± 1.03 ± 0.45 ± 1.36
SEM ± 0.52 ± 0.42 ± 0.18 ± 0.56
Range 8-11 8-11 8-9 11-15

a Endotoxin respiratory colic index scores were analyzed via three-factor analysis of variance with repeated measures on one factor.

b Serum IgG antibody measurements were analyzed via two-factor analysis of variance techniques with repeated measures on one factor.

c Mean value significantly (p < 0.05) different from control or pretreatment values.

d Mean value not significantly (p > 0.05) different from pretreatment values.

SE = Standard deviation; SEM = Standard error of the mean.

 

 

 

TABLE 3
Comparison of Combined Anorexia Time Intervals of E. coli and

Pasteurella Endotoxin-Challenged, Placebo-Treated, and Vaccinated Calves

 
Endotoxin-Challenged Calves
Parameter Placebo (control); N = 12 Vaccinate; N = 12
Mean Anorexia Time Interval (minutes)a
Mean 98.1 63.0b
SD ± 22.2 28.8
SEM ± 6.4 ± 8.31
Range 48-112 29-102

a Anorexia time interval measurements were analyzed via two-factor analysis of variance techniques with repeated measures on one factor.

b Mean value significantly (p < 0.05) different from control values.

SE = Standard deviation; SEM = Standard error of the mean.

Studies:  ENDOVAC-Bovi® in Lactating Dairy in Cows

(Summer 2007 Horton Research Center, Oxford Mountain Dairy)
Dr. David Hutcheson, Animal Nutritionist and Bovine Consultant, Animal Agricultural Consulting, Inc., P.O. Box 50367, Amarillo, TX  79159

Objective

Evaluate the effects of four bacterins’ administration on daily milk production, dry matter intake and somatic cell counts.

Introduction

Mastitis occurs in 10% to 12% of all lactating cows in the United States, with 30% to 40% of mastitis cows have inflammation due to Escherichia coli. Mastitis costs U.S. dairy producers more than $1 billion annually. Diminished milk production, discarded milk, the need for replacement cows, the decreased sale value of cows, cost of drugs, veterinary services, and additional labor all contribute to the economic losses.

Endotoxemia results from the release of endotoxins through the death of Gram-negative bacteria, such as E. coli.  This occurs during phagocytosis by udder leukocytes 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 met with varying success.

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.

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. A combination vaccine, ENDOVAC-Bovi® (IMMVAC), manufactured with the R/17 mutant and combined with an immune-potentiating adjuvant (IMMUNEPlus®), significantly reduces the devastating diseases caused by Gram-negative bacteria producing various endotoxins.

The use of ENDOVAC-Bovi® is the only core-antigen vaccine with a unique and effective immune stimulant. The patented IMMNEPlus® in combination with the mutant Re-17 bacterin protects against E. coli Mastitis and other endotoxin-mediated diseases caused by E. coli, Salmonella, Pasteurella multocida, and Pasteurella (mannheimia) hemolytica.

Procedures

Mature dairy cows at least 60 days in milk were indentified and allotted to 4 milking groups. The four groups were randomly allotted to the bacterins:

  1. ENDOVAC-Bovi®
  2. J-5
  3. J-Vac
  4. Negative Controls

Cows were randomly allotted to the treatment groups by the last 2 digits of the cows ear tags number. Three hundred and fourteen cows were allotted to four treatment groups of 80 (ENDOVAC-Bovi®), 78 (J-5), 79 (J-Vac) and 77 (Negative Control) cows per group. The cows were penned in separate pens by treatment groups. The cows were fed from 1 to 26 days with the same ration to all treatment groups.  The ration fed was the standard ration being fed at the dairy for milking cows. The treatments were administered on day 8 by manufactures label dose for all treatments. The treatments were administered as shown in Table 1.

Table 1 Treatments and Mode of Administration

Treatment Vaccine Administration
1 ENDOVAC-Bovi® 2cc (IM)
2 J-5 5cc (SC)
3 J-Vac 2cc (IM)
4 Negative Control None

 

Daily milk records and dry matter feed records were collected from each cow during the 26 day experimental period and somatic cell counts (SCC).

Results and Discussion

Table 2 shows the means and standard deviations for dry matter consumption by treatments and pre and post vaccination. There were no significant differences in dry matter consumption among the treatments either pre to or post vaccination.

Table 2. Dry matter consumption, Pre and Post Vaccination

Treatments ENDOVAC-Bovi® J-5 J-Vac Negative Control P value

Pre to Vaccination (7 days)

Mean 58.2 56.2 58.5 57.1 0.34
STD 2.8 1.9 3.4 1.8  
 

Post Vaccination (19 days)

Mean 59.3 59.3 60.1 58.5 0.50
STD 2.5 3.3 3.8 2.7  

Milk production was significantly higher for the cows to be administered J-5 and negative control pre test, Table 3. However no differences in milk production were detected after vaccination for any treatments.

Table 3. Milk Production, Pre and Post Vaccination.

Treatments ENDOVAC-Bovi® J-5 J-Vac Negative Control P value

Pre to Vaccination (7 days)

Mean 98.9b 101.2a 98.9b 100.5a 0.01
STD 1.7 1.3 1.2 1.0  
 

Post Vaccination (19 days)

Mean 99.6 100.6 99.9 100.1 0.62
STD 1.9 2.2 2.5 1.8  

Somatic Cell Counts were significantly higher for the group to be administered ENDOVAC-Bovi® than the J-Vac group pre vaccination. The J-5 was the lowest SCC but was not different from J-5 and Negative control pre vaccination. After vaccination the ENDOVAC-Bovi® group was significantly lower than other vaccination groups or negative control (Table 4).The ENDOVAC-Bovi® group proved to be the most effective control of mastitis as measured by SCC.

Table 4. Somatic Cell Counts (SCC), Pre and Post Vaccination.

Treatments ENDOVAC-Bovi® J-5 J-Vac Negative Control P value

Pre to Vaccination (7 days)

Mean 121.1a 94.9a,b 64.5b 89.4a,b 0.03
STD 48.3 41.4 10.4 9.2  
 

Post Vaccination (19 days)

Mean 81.8a 126.3b 118.7b 136.0b 0.01
STD 25.2 44.7 35.2 40.03  

Conclusions

  • ENDOVAC-Bovi® significantly lower somatic cell counts thereby decreasing mastitis in lactating dairy cow.
  • ENDOVAC-Bovi® had no effect on milk production in lactating dairy cows.
  • ENDOVAC-Bovi® had no effect on dry matter intake in the lactating dairy cow.

Studies:  Field Experience With Cross-Protective Anti-Endotoxin Antiserum in Neonatal Calves

AGRI-PRACTICE – BACTERIOLOGY

Seventeen neonatal calves comprised the ill group for Phase 1 of this study. Phase 2 was comprised of 246 head of normal neonatal calves. Twelve of the 17 ill calves received 0.8 to 1.0 ml/lb of antiserum for treatment of gram-negative diarrhea; 8 of the 12 calves received antibiotics, oral electrolytes, and anti-diarrheal treatment in addition to the antiserum; 3 of the 12 calves received antiserum only; and 1 of the 12 calves received antiserum plus oral electrolyte therapy without concomitant antibiotic therapy.
The death rate in the antiserum-treated calves was significantly (P < 0.025) lower than in the non-antiserum-treated group. No deaths subsequently occurred in the healthy, Phase 2 neonatal calves administered 0.7 ml/lb of antiserum as a precautionary measure.

 

A PRELIMINARY REPORT

Field Experience With Cross-Protective Anti-Endotoxin Antiserum in Neonatal Calves

Leroy E. Ensley, DVM
Steve M. Ensley, DVM

2nd and Prospect
Onaga, Kansas 66521

Introduction

Neonatal calves suffering from the consequences of gram-negative diarrhea have historically been treated with antimicrobials, electrolytes, and other support modes of therapy. Results of such established therapies are often frustrating because they do not neutralize the precise problem. Recent studies have confirmed that endotoxins from various sources such as Salmonella sp. and Escherichia coli may cause death in calves exhibiting the signs of diarrhea.1,2 The common denominator of all gram negative bacteria is the core lipopolysaccharide cell wall structure (endotoxin). This portion of the gram-negative cell wall is also referred to as the common-core-antigen.3
Historically, homologous antiserums produced in response to gram-negative bacterins made from organisms with complete “O” side chains have been effective in blocking specific endotoxins. These homologous antiserums have not provided cross-protective antibodies because they have contained antibodies against only one serotype. Provision of broad-spectrum protection would have required the combination of many homologous antiserums. Obviously, it would have been advantageous if a given antiserum could provide cross-protection against several gram-negative endotoxins. Therefore, the search for cross-protective immune strategies that could be used to actively immunize, treat, or passively immunize individuals against several pathogenic endotoxins is important.
The mutated Salmonella typhimurium (R17) along with the addition of an immune stimulant, toxoid (E3), has resulted in the development of a cross-protective antiserum and vaccine that are now available to veterinarians.4,5 Removal of the “O” side chains, which give the many gram-negative organisms their individual characteristics (serotype), was accomplished through mutation exposing the core-antigen of the cell wall. This core-antigen stimulates the host’s immune system to produce antibodies against it thereby providing cross-protective immunity against many of the gram-negative endotoxins.
Classification of the “O” side chains to describe the relative absence of oligosaccharides was accomplished by assigning the letters Ra through Re, with Re designating complete removal. The S. typhimurium used in the study reported here is an Re mutant (“O” side chains completely removed), while the J-5 mutant of E. coli, also cited in this report, is an Rc mutant (“O” side chains partially removed).3,5A vaccine containing such mutant bacterins used to hyperimmunize donor animals produces serum or plasma containing high levels of anti-endotoxin antibodies. These antibodies passively immunize the recipient against many gram-negative endotoxins.
Laboratory-derived evidence that antiserum provides cross-protective, passive immunity against several gram-negative bacterial endotoxins in calves1 and horses4 has recently been reported. The results of these studies suggest that calves passively immunized with plasma derived from hyperimmunized donors vaccinated with mutant gram-negative bacterins were cross-protected from various endotoxin challenges.1 Equids passively immunized with anti-core-antigen antibody antiserum and challenged under experimental conditions with a specific dose of heterologous endotoxin were also protected.4
The commercially available antiserum (Endoserum®: IMMVAC, Inc., Columbia, MO) is harvested from donor horses hyperimmunized with a mutant S. typhimurium bacterin-toxoid [Endovac-Equi®: IMMVAC, Inc.] and contains USDA standardized levels of anti-endotoxin and total IgG antibodies. Endoserum has been successfully used to treat and prevent endotoxemia in foals and horses for more than 2 years. However the effectiveness of passively immunizing calves using a mutant S. typhimurium antiserum as the source of cross-protective, anti-endotoxin antibodies has not been definitively confirmed under field conditions.
The purposes of reporting the results of this field study are:

  • To describe the results of using the cross-protective antiserum, equine origin, in neonatal calves exhibiting the signs of E. coli diarrhea/septicemia from one dairy and one beef herd; and
  • To show the results of prophylactically administering antiserum to newborn dairy and beef calves to prevent E. coli diarrhea/septicemia.

Materials and Methods

ANIMALS

The animals in Phase 1 of the study consisted of 17 head of ill neonatal calves (3 male and 6 female cross-bred calves weighing from 70-90 lbs, and 1 male and 7 Jersey calves weighing from 35-55 lbs). In Phase 2 of the study, 248 head of normal neonatal calves were studied. The group consisted of 167 cross-bred beef and 79 Jersey calves.

DIAGNOSTIC PROCEDURES

All the fecal samples collected from four ill beef and three ill Jersey calves in Phase 1 of the study tested positive for E. coli (K-99 Test Kit®: Synbiotics Corp., San Diego). All 17 of the ill calves exhibited signs of diarrhea, varying degrees of central nervous system depression, and dehydration.

ANTISERUM ADMINISTRATION

In Phase 1 of the study, 12 of the 17 ill calves were subcutaneously administered 0.8 to 1.0 ml/lb (1.75-2.2 ml/kg) of antiserum (Endoserum). Eight calves received antibiotics (Gentocin®: Schering-Plough Animal Health, Kenilworth, NJ; Naxcel®: The Upjohn Co., Animal Health Division, Kalamazoo, MI); oral electrolytes (Life-Guard®: Smith-Kline Beecham Animal Health, Exton, PA); and/or intravenous-administered electrolytes, and antidiarrhea (Deliver™: Haver/Diamond Scientific, Animal Health Division, Shawnee, KS) treatment in addition to the antiserum. Three of the calves received antiserum only, while one received antiserum, oral electrolyte therapy, and no antidiarrheal treatment. Five ill calves were treated with antibiotics, oral fluids, and antidiarrheal compounds without receiving antiserum.
In Phase 2 of the study, 246 neonatal calves received 0.7 ml/lb of antiserum at birth.

ANTIBIOTIC THERAPY

Either gentamicin (Gentocin) or ceftioufur sodium (Naxcel) was the antibiotic used for treating the calves, either concurrently with antiserum therapy or upon exhibition of signs of diarrhea following prophylactic administration of antiserum.

ELECTROLYTES

Life-Guard, lactated Ringer’s solution, and Deliver were used to treat dehydration and diarrhea.

STATISTICAL ANALYSIS

Data from Phase 1 of the study were submitted to Chi-square analysis utilizing Fischer’s exact test with the predetermined probability level of 0.05 or less used for determining significant differences.

Results

In Phase 1, the death rate in the non-antiserum-treated group was significantly (P < 0.025) higher that in the antiserum-treated group. Three (60%) of the five non-antiserum-treated calves died while none (0%) of the 12 antiserum-treated calves succumbed. Four of the acutely ill neonatal beef calves received antiserum and electrolyte therapy without antibiotics and survived.

In Phase 2 of the study, 167 cross-bred beef calves and 79 Jersey calves from the same two herds involved in Phase 1 were subsequently prophylactically administered 0.7 ml/lb of antiserum at birth. No deaths occurred in either the beef or dairy neonates that received antiserum. A few of the calves developed signs of diarrhea at various times following the prophylactic administration of the antiserum but responded quickly to routine antibiotic (Gentocin, Naxcel) therapy.
The results of both phases of the study are further illustrated in Table 1.

TABLE 1 Comparison of Escherichia coli Neonatal Calf Study Treatment Groups a

[wpcol_1half id=”” class=”” style=””]Treatment Group
[/wpcol_1half][wpcol_1quarter id=”” class=”” style=””]Survived
[/wpcol_1quarter][wpcol_1quarter_end id=”” class=”” style=””]Died
[/wpcol_1quarter_end]
[wpcol_1half id=”” class=”” style=””]Ill Calves (Phase 1)
[/wpcol_1half][wpcol_1quarter id=”” class=”” style=””] 
[/wpcol_1quarter][wpcol_1quarter_end id=”” class=”” style=””] 
[/wpcol_1quarter_end]
[wpcol_1half id=”” class=”” style=””]No antiserum given
[/wpcol_1half][wpcol_1quarter id=”” class=”” style=””]2
[/wpcol_1quarter][wpcol_1quarter_end id=”” class=”” style=””]3
[/wpcol_1quarter_end]
[wpcol_1half id=”” class=”” style=””]0.8-1.0 ml/lb body weight antiserum b
[/wpcol_1half][wpcol_1quarter id=”” class=”” style=””]12
[/wpcol_1quarter][wpcol_1quarter_end id=”” class=”” style=””]0
[/wpcol_1quarter_end]
[wpcol_1half id=”” class=”” style=””]Healthy Calves (Phase 2)
[/wpcol_1half][wpcol_1quarter id=”” class=”” style=””] 
[/wpcol_1quarter][wpcol_1quarter_end id=”” class=”” style=””] 
[/wpcol_1quarter_end]
[wpcol_1half id=”” class=”” style=””]0.7 ml/lb body weight antiserum b
[/wpcol_1half][wpcol_1quarter id=”” class=”” style=””]246
[/wpcol_1quarter][wpcol_1quarter_end id=”” class=”” style=””]0
[/wpcol_1quarter_end]
a Chi square probability = P < 0.025
b Endoserum®: IMMVAC, Inc., Columbia, MO

Discussion

The endemic colibacillosis diarrhea problems in one beef and one dairy herd were apparently brought under control with anti-endotoxin antiserum treatment. Calves with feces testing positive for pathogenic E. coli (K-99) organisms and exhibiting signs of septicemia/endotoxemia responded positively to antiserum treatment, and their subsequently calved herdmates were prevented from developing serious colibacillosis diarrhea by administration of antiserum at birth.
Neonatal calves that received antiserum prophylactically and later developed clinical signs of diarrhea responded quickly to antibiotic therapy and required very little supportive therapy. These observations suggest that E. coli endotoxins were blocked by the anti-core-antigen antibodies contained in the antiserum.
The use of antiserum as a source of cross-protective anti-endotoxin antibodies is important from the aspect that a definitive gram-negative serotype diagnosis is not required and if there are several gram-negative organisms producing endotoxins, they can be neutralized with one product. As far as the safety of equine antiserum administered to calves was concerned, there were no allergic responses observed in any of the calves. Anaphylactic shock would not be expected if repeat administrations were accomplished within 7 or 8 days following the initial administration.6 A second dose should not be administered after 7 days because the chances for anaphylactic shock will have greatly increased.6 If an allergic response occurs, administration of antiserum should be immediately ceased and epinephrine along with other modes of supportive therapy initiated.
The minimum dosage administered for treatment and control of E. coli septicemia/endotoxemia in these two groups of neonatal calves was 0.7 ml/lb. This dosage level is consistent with previously published recommendations for administering antiserum for the purposes of attenuating the clinical signs exhibited by equids challenged with a specific dose of E. coli endotoxin. Repeat administration is indicated if the ill individual does not respond or experiences a relapse of clinical signs.
In summary, the successful prevention and treatment of endemic E. coli diarrhea problems in newborn calves from two cattle herds suggested that antiserum could be used to control colibacillosis diarrhea/septicemia in other herds. Because the antiserum is cross-protective, it also suggests that the antiserum may be effective in the medical management of the devastating effects of neonatal diarrhea due to gram-negative endotoxins from various sources.

REFERENCES

1. Cullor JS, Fenwick BW, Smith BP, et al: Decreased Mortality and Severity of Infection from Salmonellosis in Calves Immunized with E. coli (Strain J5) (Abstr No. 352). Chicago, Proceedings of the 66th Annual Conference of Research Workers in Animal Disease, 1985.
2. Sprouse RF, Garner HE, Lager K: Cross-protection of Calves From E. coli and P. multocida Endotoxin Challenges Via Salmonella typhimurium Mutant Bacterin-toxoid. Agri Pract 11:29-37, 1990.
3. Tyler JW, Cullor JS, Spier SJ: Immunity Targeting Common Core Antigens of Gram-negative Bacteria. J Vet Int Med 4:17-25, 1990.
4. Garner HE, Sprouse RF, Lager K: Cross-protection of Ponies From Sublethal E. coli Endotoxemia by Salmonella typhimurium Antiserum. Eq Pract 10:10-17, 1988.
5. Sprouse RF, Garner HE, Lager K: Protection of Ponies From Heterologous and Homologous Endotoxin Challenges Via Salmonella typhimurium Bacterin-toxoid. Eq Pract 11:45-49, 1989.
6. Tizard I: Veterinary Immunology, ed. 3. Philadelphia, WB Saunders Co, 1987, p. 4.

Studies:  Gram-Negative Bacterial Protection in Beef and Dairy Production Systems

New Vaccine Technology Provides Cross-Protection With Immune Enhancement.

David F. Calabotta, Ph.D. and Thomas J. Worthington, DVM

Introduction:

In today’s intensive beef and dairy production systems, one must recognize and address the potential negative impact of management practices which stress the animal. These stresses can predispose animals to gram-negative, opportunistic challenges resulting in severe losses in performance, morbidity and death. Housing animals under close quarters, extreme climatic conditions, abrupt nutritional changes, feed toxins, and other typical stresses associated with shipping of animals to the feedlot, can all result in a predisposition to opportunistic diseases. In the intensive dairy system, high producing cows may be more susceptible to gram-negative associated mastitis and/or salmonella and E.Coli infections. Opportunistic diseases may also be initially manifested as viral challenges. In the majority of these viral disease situations, the establishment of secondary gram negative infections and associated endotoxemias may result in reduced performance and death.

New Technology Provides Economically Viable Options For Gram-Negative Cross-Protection:

To prevent the above described disease conditions, the producer has in the past relied upon vaccination programs to protect against viral and bacterial infections and the use of antibiotic programs to ward off and/or eliminate bacterial insults already established. However, to effectively protect against all viral and bacterial challenges and the many thousands of different bacterial serotypes, one would have to literally administer thousands of vaccinations and utilize a very intensive antibiotic program. Even under the above described intensive health management practices, complete protection would not be assured due to a myrad of bacterial culprits and constantly mutating serotypes. However, new technology has been recently invented and developed which may in fact provide complete protection from the typical gram negative secondary bacterial infections associated with viral disease development while stimulating the overall protection fighting capacity of the animal’s immune system.

The concept involves the development of a proprietary, recombinantly produced Re-17 mutant bacterin coupled with an immune stimulation system comprised of an antitoxoid antigen. This vaccine cocktail stimulates and jump starts the animal’s immune system such that both humoral (antibody production) and cellurlar (cellular killing) immunity may be successfully achieved, while cross protecting the animal against essentially all gram negative diseases.

The production of the Re-17 mutant bacterin involves the removal of the bacteria’s oligosacharride side chains which results in the production of a bacteria with a naked outer core. (Figure 1.)

In a non-mutant bacteria, the antigenic response is normally targeted specifically at the O-side chains, and while this process results in specific protection, the protection is only limited to a specific bacterial serotype. By producing a mutant bacterin devoid of the O-side chains, scientists at IMMVAC, Incorporated, in conjunction with scientists at the University Of Missouri School of Human Medicine and The School Of Veterinary Medicine, have succeeded in removing those antigenic sites responsible for a specific antibody production. The result is a bacterin which contains a naked core (cell wall) which is common to all gram negative bacteria and their many thousands of serotypes. The antigenicity of this recombinantly produced bacterin is now focused upon sites within the naked core which results in the bacterin stimulating universal cross-protection against essentially all gram negative bacteria and their serotypes. In addition, by combining an immune stimulation component to the system, the animal’s immune system is enhanced and more efficient in providing both humoral and cellular immunity. Finally, since the system is comprised of an inactivated or killed vaccine, the herd veterinarian may administer herd health antibiotic programs, without compromising immunity development to the vaccine. Thus, maximum herd health management flexibility is achieved.

Scientific Evaluations Confirm Both Physiological Potency and Practical Effectiveness Of ENDOVAC-Bovi® With IMMUNE Plus:

Figure 2. depicts the effectiveness of this novel vaccination system, particularly the E3 immune system enhancing component in stimulating the overall immune response.

Insert Figure 2

Peripheral circulation of both B and T lymphocytes are enhanced with the bacterin plus anti-toxoid (E3 stimulation is administered). As predicted, antibody production is also increased (Figure 3).

Insert Figure 3

Using this novel vaccination system, a producer can achieve herd health protection as never before, while delivering significant profits to his bottomline. In controlled field studies1 conducted at a major beef feedlot in the southwestern United States, 351 beef steers receiving ENDOVAC-Bovi were compared to a similar control group receiving their normal vaccination and herd health management schedule. (Figure 4.)

Insert Figure 4

summarizes the results of the study. Total weight gain of the vaccinates was increased by 5183 lbs, although this was not statistically significant. In addition, morbidity in the herd was significantly reduced as evidenced by a significant reduction in 1st and 2nd pulled calves requiring treatment. Of the five respiratory deaths that occurred due to bovine respiratory disease complex (BRDC), only one was a vaccinate animal. When the producer evaluated the cost of the herd health program associated with sick calves and pulled calves, it was concluded that herd health costs were significantly reduced in steers receiving ENDOVAC-Bovi compared to the control group receiving their normal vaccination program and herd health management program. Total cost improvement was $12.50/head in those animals who received ENDOVAC-Bovi. These results were confirmed by Kennedy2, 1995 (Figure 5).

Insert Figure 5

He concluded that the Re-17 mutant Salmonella typhimurium bacterin toxoid significantly decreased the respiratory disease morbidity and severity in vaccinated steers. Also, fewer deaths occurred (p = 0.11), fewer first and second pulls requiring medical treatment (p<0.005) and overall herd health costs were reduced (p<0.005) in the vaccinated group.

In studies conducted in dairy cows3, significant reduction in mastitis was observed in those cows who received ENDOVAC-Bovi. In addition recurrence of mastitis in specific cows who had previously suffered a bout of mastitis was significantly reduced in those animals who had received ENDOVAC-Bovi.

In calves headed for the feedlot and/or replacement heifers4, those animals who received an early vaccination with ENDOVAC-Bovi, had significantly fewer bouts with gram-negative associated bacterial challenges and were overall healthier as evidenced by improved performance and a significant reduction in mortality and morbidity. In addition, significant reductions in herd health management costs were noted.

Recommended Vaccination Schedules:

Beef Cows, Dairy Cows & Their Calves:

For maximum, efficient protection, it is recommended that all beef cows be vaccinated 7-10 days prior to shipping to a feedlot with a 2 ml dose of ENDOVAC-Bovi. If logistics permit, a booster 10-14 days following the first injection is advised, however very good results and herd protection have been achieved with only one vaccination. Your herd veterinarian should be consulted to assist you in determining the best course of action.

In pregnant cows (beef or dairy), it is recommended that the dam be vaccinated with a 2 ml dose of ENDOVAC-Bovi 4 weeks prior to parturition with a 2 ml booster dose being administered one week prior to parturition. This practice will provide maximum immunity to the dam while providing significant passive immunity to the new-born calf. In the case of the dairy cow, significantly less mastitis will result.

The calf should be vaccinated with the following vaccination program4

* Day 1: 1 ml injection IM*
* Day 4: 1 ml injection IM*
* Day 10: 1 ml injection IM*

An additional injection is suggested to provide protection against Pasteurella moltocida and hemolytica:

* 3-5 weeks: 2 ml injection IM*

(*intramuscularly)

Summary and Conclusions:

In today’s intensive beef and dairy production systems, the use of a new, proprietary, universal, cross-protective vaccine, effective against all gram negative bacteria may significantly protect the beef cow from secondary gram-negative challenges associated with environmental stress conditions and/or viral disease situations. When used as directed, the producer can save significant dollars by significantly reducing performance and mortality associated losses resulting from the above described disease challenges. In dairy animals, significant reduction in gram-negative associated diseases, including mastitis related challenges have been demonstrated when animals are properly vaccinated with ENDOVAC-Bovi. When used as directed by the manufacturer, the beef or dairy producer can achieve maximum health protection in their cows, calves and feedlot cattle while saving significant dollars on herd health costs and increasing profits through performance improvements derived from this vaccination system.

About the Authors:

Dr. Thomas J. Worthington is a doctor of veterinary medicine and owner and president of Chino Corona Veterinary Services, a consultant group serving the beef and dairy cattle industry in the western United States. Dr. David F. Calabotta has a Ph.D. in animal science and is the owner and president of Anitech, Inc., a business development firm specializing in the late stage commercial development of emerging, proprietary animal technologies.

References Cited:

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4. Worthington, Thomas J. 1996. The Rationale Of Mass Treatment Of Gram-Negative Scours In Large Calf Ranches. American Association of Bovine Practitioners 29th Annual Conference: September 12-14; San Diego, California.