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


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.



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


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


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.


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.


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.


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.


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


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.


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_1half id=”” class=”” style=””]Ill Calves (Phase 1)
[/wpcol_1half][wpcol_1quarter id=”” class=”” style=””] 
[/wpcol_1quarter][wpcol_1quarter_end id=”” class=”” style=””] 
[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_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_1half id=”” class=”” style=””]Healthy Calves (Phase 2)
[/wpcol_1half][wpcol_1quarter id=”” class=”” style=””] 
[/wpcol_1quarter][wpcol_1quarter_end id=”” class=”” style=””] 
[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
a Chi square probability = P < 0.025
b Endoserum®: IMMVAC, Inc., Columbia, MO


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.


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.