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A Comparison of Two Commercially Available Escherichia coli J5 Vaccines against E. coli Intramammary Challenge1
A Comparison of Two Commercially Available Escherichia coli J5 Vaccines against E. coli Intramammary Challenge1 G. M. Tomita,† C. H. Ray,† S. C. Nickerson,† W. E. Owens,† and G. F. Gallo‡ †Hill Farm Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, 11959 Hwy 9, Homer 71040 ‡Merial Limited, 115 Transtech Dr. Athens, GA 30601
ABSTRACT The efficacy of two commercially available Escherichia coli J5 bacterins was investigated. Jersey cows were randomly assigned to one of three treatment groups: 1) unvaccinated controls, 2) vaccinated with JⴢVAC (Merial Limited, Athens, GA), and 3) vaccinated with J5 bacterin. All cows were vaccinated at drying off and at 2 wk before anticipated calving. Cows that were vaccinated with the J5 bacterin also received a third immunization at calving. One quarter of each cow was challenged with approximately 64 cfu of E. coli at 14 to 30 d postcalving. Immunization by either vaccine did not influence the severity of coliform mastitis; however, the mean number of colony-forming units of E. coli recovered from challenged quarters was significantly lower for immunized cows than for control cows at 144 h postchallenge. Serum and mammary secretion immunoglobulin (Ig)G, IgG1, and IgG2 titers against E. coli J5 whole-cell antigens were enhanced in vaccinated cows. Serum and mammary secretion IgM were not different among treatment groups. Somatic cell counts in milk from challenged quarters, rectal temperatures, and the clinical status of cows following intramammary challenge were not different among treatment groups. (Key words: Escherichia coli J5, immunization, intramammary challenge, mastitis) Abbreviation key: LPS = lipopolysaccharide. INTRODUCTION Mastitis continues to be the most economically important disease in the dairy industry (2). Coliform mas-
Received September 21, 1999. Accepted April 12, 2000. Corresponding author: S. C. Nickerson; e-mail: snickerson@agctr. lsu.edu. 1 Approved for publication by the director of the Louisiana State University Agricultural Experiment Station as Manuscript Number 99-08-0613. 2000 J Dairy Sci 83:2276–2281
titis presents a unique problem to the dairy producer despite advances in herd health management practices. Postmilking teat dipping and dry cow antibiotic therapy are ineffective in the control of coliform mastitis due to continuous teat end exposure and the ubiquity of coliform pathogens in the environment of cows (11). Active immunization against coliform bacteria appears to control episodes of clinical coliform mastitis if employed in conjunction with a well-managed herd health program (4, 6). The sudden, acute symptoms of clinical coliform mastitis have been attributed to the presence of lipopolysaccharide (LPS) on the outer cell wall of coliform bacteria (1). The core and lipid A regions of LPS possess antigenic homology among coliform pathogens and have been targeted as vaccine antigens. A mutant strain of Escherichia coli O111:B4 (J5) has a unique characteristic in which the core and lipid A antigens of LPS are exposed, and immunization with E. coli J5 produces antibodies that are cross reactive with other coliform pathogens (14, 15). Results from challenge (7, 8, 12) and field (4, 6) trials have shown that immunization with an E. coli J5 bacterin increases antibody titers to E. coli J5 core antigens in serum and mammary secretions. Naturally enhanced antibody titers to E. coli J5 were associated with a decreased risk of developing clinical coliform mastitis (13), and immunization with E. coli J5 reduced the severity and lowered the rates of clinical coliform mastitis (4, 6, 7, 8). Two commercially produced vaccines based on E. coli J5 bacterins are currently available. The manufacturer of one product suggests the administration of two doses (JⴢVAC, Merial Limited, Athens, GA), while the manufacturer of the other product suggests three doses (J5 bacterin, Escherichia coli Bacterin J5 Strain, Pharmacia & Upjohn, Kalamazoo, MI). The objective of the present study was to determine the efficacy of the two vaccines against experimental intramammary challenge with E. coli.
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MATERIALS AND METHODS Vaccination Schedule Jersey cows from the Hill Farm Research Station dairy herd were assigned to one of three treatment groups and blocked by age, stage of lactation, and parity. Each block included one cow from the following treatment groups: nonvaccinated controls (n = 8), vaccinated subcutaneously in the neck with JⴢVAC (n = 8), and vaccinated subcutaneously in the neck with J5 bacterin (n = 8). Immunized cows were vaccinated at drying off (d 0) and received a secondary vaccination approximately 2 wk before anticipated calving (d 45). Cows immunized with the J5 bacterin received a tertiary vaccination at calving. Both vaccines were administered as recommended by the manufacturers. Control cows did not receive an injection at all. The general health, site of immunization, and rectal temperatures of vaccinated cows were monitored immediately before immunization and at 12, 24, 48, and 72 h following each vaccine administration.
Figure 1. Least-squares means for log10 colony-forming units of Escherichia coli recovered from challenged quarters. Quarters were challenged at 0 h. Different letters (a, b) within hours indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Lines above bars represent standard errors of the means.
Intramammary Challenge and Milk Samples One uninfected quarter of each cow within a block was infused with 64.1 ± 4.2 cfu of live E. coli at the stationary growth phase suspended in 1 ml of sterile PBS (pH 7.2). The challenge dose was prepared as described by Hogan et al. (8). All cows were challenged 14 to 30 d postcalving at 5 to 6 h after the morning milking. The challenge strain (E. coli 727) was previously isolated from a clinical mastitic quarter (7). Aseptic milk samples from the challenged quarters were collected (9) immediately before challenge and at 4, 8, 12, 16, 20, 24, 48, 72, 96, 120, and 144 h postchallenge. The numbers of colony-forming units in the challenge dose and colony-forming units per milk sample were determined by MacConkey agar (Becton Dickinson and Co., Cockeysville, MD) pour plates. The SCC, rectal temperatures, and clinical status of cows were used to determine severity of clinical mastitis. Milk sample SCC was determined with a Fossomatic cell counter (A/N Foss Electric, Hillerød, Denmark). Rectal temperatures and clinical status of cows were also monitored at the intervals mentioned previously. The clinical status of cows was scored on a scale of 1 to 5, where 1 = normal milk and quarter, 2 = questionable milk and normal quarter, 3 = abnormal milk and normal quarter, 4 = abnormal milk and swollen quarter, and 5 = abnormal milk, swollen quarter, and pyrexia (8). Antibody Titers Antibody titers of serum and mammary secretions to E. coli J5 whole-cell antigens were determined by
ELISA (14). Samples were collected on d 0, 21, and 45 of the dry period, at calving, and on d 14, 21, 30, and 45 postcalving. The following isotypes were assayed: IgG, IgG1, IgG2, and IgM. Statistical Analysis Comparisons of colony-forming units, SCC, rectal temperatures, clinical status scores, and antibody titers among blocks and among treatment groups were analyzed using the mixed models procedure for measures repeated across time (3). Colony-forming units and SCC data were transformed to log10, and the reciprocal of end point antibody titer dilution was transformed to log2. Data were analyzed using the general linear models procedure of SAS and tested by least squares ANOVA (10). RESULTS Immunization with the J5 bacterin or JⴢVAC did not adversely affect the general health of cows. Minimal swelling (<4.5 cm) was observed at the injection site, and systemic response to immunization was not evident. The challenge dose of E. coli among blocks was not different (P > 0.05). The mean number of bacteria infused into one quarter of all cows was 64.1 ± 4.2 cfu (59 to 72 cfu). The number of colony-forming units recovered from challenged quarters of all cows peaked at 12 h postchallenge and began to decrease by 16 h (Figure 1). At 72 h postchallenge, the number of colonyJournal of Dairy Science Vol. 83, No. 10, 2000
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forming units recovered from control cows remained constant (approximately 1.5 log10 cfu/ml), while the number of colony-forming units recovered from immunized cows continued to decline. Compared with control cows, immunized cows began to exhibit a significant reduction (P < 0.05) in the number of colony-forming units at 144 h postchallenge. The number of colonyforming units at 144 h postchallenge was not different between cows immunized with JⴢVAC compared with those immunized with J5 bacterin. Somatic cell counts, rectal temperatures, and clinical status scores among treatment groups and among blocks were not different (P > 0.05; data not shown). A typical response to induced coliform mastitis was observed. Milk SCC peaked by 12 h postchallenge and remained elevated (>106/ml) throughout the study. Rectal temperatures began to increase 8 h following intramammary challenge, peaked at 16 h, and returned to prechallenge values by 24 h. The clinical status score of cows reflected the SCC and rectal temperature trends following intramammary challenge. A score of 2 or 3 (abnormal milk, normal quarter) was observed in all treatment groups by 12 h postchallenge, and a clinical status score of 4 or 5 (abnormal milk and swollen quarter, or pyrexia) was observed at 16, 20, and 24 h postchallenge. All cows had a clinical status score of 3 from 48 to 144 h postchallenge. Similar trends in serum IgG (Figure 2), IgG1 (Figure 3), and IgG2 (Figure 4) antibody titers to E. coli J5
Figure 2. Least-squares means for log2 serum IgG antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b, c) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Asterisks indicate a difference (P < 0.05) within treatment groups compared with values on the day of dry-off (D-0). Cows were immunized with J5 bacterin on D-0, D+45, and at calving (C+0) or immunized with JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving. Journal of Dairy Science Vol. 83, No. 10, 2000
Figure 3. Least-squares means for log2 serum IgG1 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b, c) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with the J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bar; n = 8). Asterisks indicate a difference (P < 0.05) within treatment groups compared with values on the day of dry-off (D-0). Cows were immunized with J5 bacterin on D-0, D+45, and at calving (C+0) or immunized with JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving.
whole-cell antigens were observed. Immunized cows had higher (P < 0.05) antibody titers on d 21 and 45 after dry-off, at calving, and on d 14, 21, 30, and 45 after calving compared with control cows. Antibody titers of immunized cows were also elevated (P < 0.05) on d 21 after dry-off and on d 21, 30, and 45 after calving compared with titers on d 0. Cows immunized with J5 bacterin had higher IgG titers (P < 0.05) on d 30 and 45 after calving and higher IgG1 titers (P < 0.05) on d 30 after calving compared with JⴢVAC immunized and control cows. Serum IgG, IgG1, and IgG2 titers of control cows, and serum IgM titers of all cows (data not shown) remained unchanged throughout the study. Whey IgG, IgG1, and IgG2 titers to E. coli J5 wholecell antigens are shown in Figures 5, 6, and 7, respectively. Immunization with the J5 bacterin enhanced (P < 0.05) the IgG1 immune response on d 14, 21, 30, and 45 after calving, and enhanced the IgG2 immune response on d 21 and 45 after dry-off, at calving, and on d 21, 30, and 45 after calving compared with control cows. The JⴢVAC immunization elevated (P < 0.05) IgG1 titers at calving, and on d 14, 21, and 45 after calving, and an increase (P < 0.05) in IgG2 titers was also observed at calving compared with control cows. Whey IgM titers between control and vaccinated cows were not different (P > 0.05; data not shown).
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Figure 4. Least-squares means for log2 serum IgG2 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with the J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Asterisks indicate a difference (P < 0.05) within treatment groups compared with values on the day of dry-off (D-0). Cows were immunized with the J5 bacterin on D-0, D+45, and at calving (C+0) or immunized with JⴢVAC on D0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving.
Figure 6. Least-squares means for log2 whey IgG1 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Cows were immunized with the J5 bacterin on D-0, D+45, and at calving (C+0), and immunized with JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving.
Figure 5. Least-squares means for log2 whey IgG antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with the J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Cows were immunized with the J5 bacterin on D-0, D+45, and at calving (C+0) and immunized with the JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving.
Figure 7. Least-squares means for log2 whey IgG2 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with the J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Cows were immunized with the J5 bacterin on D-0, D+45, and at calving (C+0) or immunized with JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving. Journal of Dairy Science Vol. 83, No. 10, 2000
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DISCUSSION Previous studies (4, 6, 8) that tested the efficacy of E. coli J5 vaccines have shown that immunization effectively reduced the incidence and severity of clinical coliform mastitis. The timing of vaccine administration has been based on the period of greatest risk of acquiring coliform mastitis, which has been shown to occur during the early dry period, the late dry period, and at calving (11). The commercially available J5 bacterin employed in this study recommends an immunization protocol that coincides with the periods of susceptibility mentioned above; e.g., at dry-off, mid dry period, and at calving. A recent study (12) with JⴢVAC showed that two immunizations (at dry-off and 45 d later) evoked an immune response, which elevated antibody titers and enhanced bacterial clearance from the mammary gland following intramammary challenge. In the present study, the J5 bacterin or JⴢVAC immunization increased serum and whey antibody titers over those of controls. These results concur with previous studies that investigated vaccines based on E. coli J5 (6, 7, 8, 12). One study (7) demonstrated that challenging one quarter with approximately 60 cfu of E. coli 727 produced a mild case of clinical coliform mastitis. In another study (12), two quarters of each cow were challenged with approximately 60 cfu of E. coli 727, which resulted in moderate to very severe clinical mastitis despite immunization. Cows in the current study were challenged in a single quarter to evaluate the efficacy of immunization on a more moderate infection. However, infusion of approximately 64 cfu of E. coli 727 into one quarter also induced moderate to severe clinical mastitis. The severity of infection observed in the current study may have overwhelmed the immune system of vaccinated cows, and, therefore, masked the possible effects of immunization on clinical symptoms. This phenomenon was also noted in a previous study (12). This occurred despite the reduction in colony-forming units observed in vaccinated cows. A reduction in the severity and incidence of clinical coliform mastitis has been attributed to enhanced bacterial clearance from challenged quarters and neutralization of LPS in association with elevated antibody titers to LPS core antigens (7, 8, 13). This association was observed in this study, in which immunized cows had a significantly higher rate of bacterial clearance at 144 h postchallenge than did control cows; this coincided with enhanced serum and whey IgG titers at 30 and 45 d postcalving. However, immunization with the J5 bacterin or JⴢVAC did not influence severity of clinical symptoms following intramammary challenge. This observation was noted despite the elevated presence of Journal of Dairy Science Vol. 83, No. 10, 2000
IgG1 and IgG2, which serve as opsonins of phagocytosis (5) and to neutralize endotoxin (16), respectively. A significant increase in whey IgG2 was observed at 21, 30, and 45 d postcalving in cows immunized with J5 bacterin; however, this enhanced level had no apparent influence on the effect of endotoxemia. In the present study, a marked increase in serum IgG, IgG1, and IgG2 titers was observed following the primary immunization at drying off. An immune response to the secondary immunization was evident at calving, and IgG titers remained elevated for the duration of the study. A tertiary immunization at calving with the J5 bacterin did not significantly increase antibody titers above control or JⴢVAC immunized cows until 30 to 45 d later, a period following intramammary challenge. Immunization with J5 bacterin or JⴢVAC also elevated whey IgG antibody titers to E. coli J5 whole-cell antigens. Whey IgG2 titers during the dry period (d 21 and 45) and lactation (d 21, 30, and 45 postcalving) were significantly enhanced by J5 bacterin immunization, while JⴢVAC immunized cows had elevated IgG1 titers at calving. This observation is in agreement with previously published results (12). Cows with preexisting elevated antibody titers to E. coli J5 have a reduced risk of developing clinical coliform mastitis (13). Therefore, results from this study suggest that immunization with J5 bacterin or JⴢVAC might have reduced the risk of acquiring coliform mastitis during the dry period. In conclusion, the current study suggests that immunization with the JⴢVAC (two doses) or J5 bacterin (three doses) elicits a similar immune response to E. coli J5 whole cell antigens. Cows receiving the third vaccination of J5 bacterin exhibited higher antibody titers following intramammary challenge, although the benefit of the tertiary immunization with respect to clinical symptoms and bacterial clearance from the mammary gland was not evident in this study. Severity of coliform mastitis was not influenced by either vaccine. However, immunization with the JⴢVAC or J5 bacterin enhanced bacterial clearance from challenged quarters and elevated antibody titers to E. coli J5. REFERENCES 1 Bauman, H., and J. Gauldie. 1994. The acute phase response. Immunol. Today 15:74–80. 2 DeGraves, F. J., and J. Fetrow. 1991. Partial budget analysis of vaccinating dairy cattle against coliform mastitis with an Escherichia coli J5 vaccine. J. Am. Vet. Med. Assoc. 199:451–455. 3 Gill, J. L., and H. D. Hafs. 1971. Analysis of repeated measurement of animals. J. Anim. Sci. 33:331–336. 4 Gonzalez, R. N., J. S. Cullor, D. E. Jasper, T. B. Farver, R. B. Bushnell, and M. N. Oliver. 1989. Prevention of clinical coliform mastitis in dairy cows by a mutant Escherichia coli vaccine. Can. J. Vet. Res. 53:301–305.
IMMUNIZATION AGAINST COLIFORM MASTITIS 5 Hill, A .W., D.J.S. Heneghan, T. R. Field, and M. R. Williams. 1983. Increase in specific opsonic activity in bovine milk following experimental Escherichia coli mastitis. Res. Vet. Sci. 35:222–227. 6 Hogan, J. S., K. L. Smith, D. A. Todhunter, and P. S. Schoenberger. 1992. Field trial to determine efficacy of an Escherichia coli J5 mastitis vaccine. J. Dairy Sci. 75:78–84. 7 Hogan, J. S., W. P. Weiss, K. L. Smith, D. A. Todhunter, P. S. Schoenberger, and L. M. Sordillo. 1995. Effect of an Escherichia coli J5 vaccine on mild clinical coliform mastitis. J. Dairy Sci. 78:285–290. 8 Hogan, J. S., W. P. Weiss, D. A. Todhunter, K. L. Smith, and P. S. Schoenberger. 1992. Efficacy of an Escherichia coli J5 mastitis vaccine in an experimental challenge trial. J. Dairy Sci. 75:415–422. 9 National Mastitis Council. 1987. Current Concepts of Bovine Mastitis. 3rd ed. Natl. Mastitis Counc., Inc., Arlington, VA. 10 SAS/STAT User’s Guide, Release 6.03. 1988. SAS Inst. Inc., Cary, NC.
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11 Smith, K. L., D. A. Todhunter, and P. S. Schoenberger. 1985. Environmental mastitis: cause, prevalence, prevention. J. Dairy Sci. 68:1531–1553. 12 Tomita, G. M., S. C. Nickerson, W. E. Owens, and B. Wren. 1998. Influence of route of vaccine administration against experimental intramammary infection caused by Escherichia coli. J. Dairy Sci. 81:2159–2164. 13 Tyler, J. W., J. S. Cullor, B. I. Osburn, R. B. Bushnell, and B. W. Fenwick. 1988. Relationship between serologic recognition of Escherichia coli O111:B4 (J5) and clinical coliform mastitis in cattle. Am. J. Vet. Res. 49:1950–1954. 14 Tyler, J., H. Spears, J. Cullor, and W. Smith. 1991. Antigenic homology among Gram-negative organisms isolated from cattle with clinical mastitis. J. Dairy Sci. 74:1235–1242. 15 Tyler, J. W., H. Spears, and R. Nelson. 1992. Antigenic homology of endotoxin with a coliform mastitis vaccine strain, Escherichia coli O111:B4 (J5). J. Dairy Sci. 75:1821–1825. 16 Zeigler, E. J., H. Douglas, J. E. Sherman, C. E. Davis, and A. I. Braude. 1973. Treatment of E. coli and Klebsiella bacteremia in agranulocytic animals with antiserum to a udp-gal epimerasedeficient mutant. J. Immunol. 111:433–439.
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ABSTRACT The efficacy of two commercially available Escherichia coli J5 bacterins was investigated. Jersey cows were randomly assigned to one of three treatment groups: 1) unvaccinated controls, 2) vaccinated with JⴢVAC (Merial Limited, Athens, GA), and 3) vaccinated with J5 bacterin. All cows were vaccinated at drying off and at 2 wk before anticipated calving. Cows that were vaccinated with the J5 bacterin also received a third immunization at calving. One quarter of each cow was challenged with approximately 64 cfu of E. coli at 14 to 30 d postcalving. Immunization by either vaccine did not influence the severity of coliform mastitis; however, the mean number of colony-forming units of E. coli recovered from challenged quarters was significantly lower for immunized cows than for control cows at 144 h postchallenge. Serum and mammary secretion immunoglobulin (Ig)G, IgG1, and IgG2 titers against E. coli J5 whole-cell antigens were enhanced in vaccinated cows. Serum and mammary secretion IgM were not different among treatment groups. Somatic cell counts in milk from challenged quarters, rectal temperatures, and the clinical status of cows following intramammary challenge were not different among treatment groups. (Key words: Escherichia coli J5, immunization, intramammary challenge, mastitis) Abbreviation key: LPS = lipopolysaccharide. INTRODUCTION Mastitis continues to be the most economically important disease in the dairy industry (2). Coliform mas-
Received September 21, 1999. Accepted April 12, 2000. Corresponding author: S. C. Nickerson; e-mail: snickerson@agctr. lsu.edu. 1 Approved for publication by the director of the Louisiana State University Agricultural Experiment Station as Manuscript Number 99-08-0613. 2000 J Dairy Sci 83:2276–2281
titis presents a unique problem to the dairy producer despite advances in herd health management practices. Postmilking teat dipping and dry cow antibiotic therapy are ineffective in the control of coliform mastitis due to continuous teat end exposure and the ubiquity of coliform pathogens in the environment of cows (11). Active immunization against coliform bacteria appears to control episodes of clinical coliform mastitis if employed in conjunction with a well-managed herd health program (4, 6). The sudden, acute symptoms of clinical coliform mastitis have been attributed to the presence of lipopolysaccharide (LPS) on the outer cell wall of coliform bacteria (1). The core and lipid A regions of LPS possess antigenic homology among coliform pathogens and have been targeted as vaccine antigens. A mutant strain of Escherichia coli O111:B4 (J5) has a unique characteristic in which the core and lipid A antigens of LPS are exposed, and immunization with E. coli J5 produces antibodies that are cross reactive with other coliform pathogens (14, 15). Results from challenge (7, 8, 12) and field (4, 6) trials have shown that immunization with an E. coli J5 bacterin increases antibody titers to E. coli J5 core antigens in serum and mammary secretions. Naturally enhanced antibody titers to E. coli J5 were associated with a decreased risk of developing clinical coliform mastitis (13), and immunization with E. coli J5 reduced the severity and lowered the rates of clinical coliform mastitis (4, 6, 7, 8). Two commercially produced vaccines based on E. coli J5 bacterins are currently available. The manufacturer of one product suggests the administration of two doses (JⴢVAC, Merial Limited, Athens, GA), while the manufacturer of the other product suggests three doses (J5 bacterin, Escherichia coli Bacterin J5 Strain, Pharmacia & Upjohn, Kalamazoo, MI). The objective of the present study was to determine the efficacy of the two vaccines against experimental intramammary challenge with E. coli.
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MATERIALS AND METHODS Vaccination Schedule Jersey cows from the Hill Farm Research Station dairy herd were assigned to one of three treatment groups and blocked by age, stage of lactation, and parity. Each block included one cow from the following treatment groups: nonvaccinated controls (n = 8), vaccinated subcutaneously in the neck with JⴢVAC (n = 8), and vaccinated subcutaneously in the neck with J5 bacterin (n = 8). Immunized cows were vaccinated at drying off (d 0) and received a secondary vaccination approximately 2 wk before anticipated calving (d 45). Cows immunized with the J5 bacterin received a tertiary vaccination at calving. Both vaccines were administered as recommended by the manufacturers. Control cows did not receive an injection at all. The general health, site of immunization, and rectal temperatures of vaccinated cows were monitored immediately before immunization and at 12, 24, 48, and 72 h following each vaccine administration.
Figure 1. Least-squares means for log10 colony-forming units of Escherichia coli recovered from challenged quarters. Quarters were challenged at 0 h. Different letters (a, b) within hours indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Lines above bars represent standard errors of the means.
Intramammary Challenge and Milk Samples One uninfected quarter of each cow within a block was infused with 64.1 ± 4.2 cfu of live E. coli at the stationary growth phase suspended in 1 ml of sterile PBS (pH 7.2). The challenge dose was prepared as described by Hogan et al. (8). All cows were challenged 14 to 30 d postcalving at 5 to 6 h after the morning milking. The challenge strain (E. coli 727) was previously isolated from a clinical mastitic quarter (7). Aseptic milk samples from the challenged quarters were collected (9) immediately before challenge and at 4, 8, 12, 16, 20, 24, 48, 72, 96, 120, and 144 h postchallenge. The numbers of colony-forming units in the challenge dose and colony-forming units per milk sample were determined by MacConkey agar (Becton Dickinson and Co., Cockeysville, MD) pour plates. The SCC, rectal temperatures, and clinical status of cows were used to determine severity of clinical mastitis. Milk sample SCC was determined with a Fossomatic cell counter (A/N Foss Electric, Hillerød, Denmark). Rectal temperatures and clinical status of cows were also monitored at the intervals mentioned previously. The clinical status of cows was scored on a scale of 1 to 5, where 1 = normal milk and quarter, 2 = questionable milk and normal quarter, 3 = abnormal milk and normal quarter, 4 = abnormal milk and swollen quarter, and 5 = abnormal milk, swollen quarter, and pyrexia (8). Antibody Titers Antibody titers of serum and mammary secretions to E. coli J5 whole-cell antigens were determined by
ELISA (14). Samples were collected on d 0, 21, and 45 of the dry period, at calving, and on d 14, 21, 30, and 45 postcalving. The following isotypes were assayed: IgG, IgG1, IgG2, and IgM. Statistical Analysis Comparisons of colony-forming units, SCC, rectal temperatures, clinical status scores, and antibody titers among blocks and among treatment groups were analyzed using the mixed models procedure for measures repeated across time (3). Colony-forming units and SCC data were transformed to log10, and the reciprocal of end point antibody titer dilution was transformed to log2. Data were analyzed using the general linear models procedure of SAS and tested by least squares ANOVA (10). RESULTS Immunization with the J5 bacterin or JⴢVAC did not adversely affect the general health of cows. Minimal swelling (<4.5 cm) was observed at the injection site, and systemic response to immunization was not evident. The challenge dose of E. coli among blocks was not different (P > 0.05). The mean number of bacteria infused into one quarter of all cows was 64.1 ± 4.2 cfu (59 to 72 cfu). The number of colony-forming units recovered from challenged quarters of all cows peaked at 12 h postchallenge and began to decrease by 16 h (Figure 1). At 72 h postchallenge, the number of colonyJournal of Dairy Science Vol. 83, No. 10, 2000
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forming units recovered from control cows remained constant (approximately 1.5 log10 cfu/ml), while the number of colony-forming units recovered from immunized cows continued to decline. Compared with control cows, immunized cows began to exhibit a significant reduction (P < 0.05) in the number of colony-forming units at 144 h postchallenge. The number of colonyforming units at 144 h postchallenge was not different between cows immunized with JⴢVAC compared with those immunized with J5 bacterin. Somatic cell counts, rectal temperatures, and clinical status scores among treatment groups and among blocks were not different (P > 0.05; data not shown). A typical response to induced coliform mastitis was observed. Milk SCC peaked by 12 h postchallenge and remained elevated (>106/ml) throughout the study. Rectal temperatures began to increase 8 h following intramammary challenge, peaked at 16 h, and returned to prechallenge values by 24 h. The clinical status score of cows reflected the SCC and rectal temperature trends following intramammary challenge. A score of 2 or 3 (abnormal milk, normal quarter) was observed in all treatment groups by 12 h postchallenge, and a clinical status score of 4 or 5 (abnormal milk and swollen quarter, or pyrexia) was observed at 16, 20, and 24 h postchallenge. All cows had a clinical status score of 3 from 48 to 144 h postchallenge. Similar trends in serum IgG (Figure 2), IgG1 (Figure 3), and IgG2 (Figure 4) antibody titers to E. coli J5
Figure 2. Least-squares means for log2 serum IgG antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b, c) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Asterisks indicate a difference (P < 0.05) within treatment groups compared with values on the day of dry-off (D-0). Cows were immunized with J5 bacterin on D-0, D+45, and at calving (C+0) or immunized with JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving. Journal of Dairy Science Vol. 83, No. 10, 2000
Figure 3. Least-squares means for log2 serum IgG1 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b, c) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with the J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bar; n = 8). Asterisks indicate a difference (P < 0.05) within treatment groups compared with values on the day of dry-off (D-0). Cows were immunized with J5 bacterin on D-0, D+45, and at calving (C+0) or immunized with JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving.
whole-cell antigens were observed. Immunized cows had higher (P < 0.05) antibody titers on d 21 and 45 after dry-off, at calving, and on d 14, 21, 30, and 45 after calving compared with control cows. Antibody titers of immunized cows were also elevated (P < 0.05) on d 21 after dry-off and on d 21, 30, and 45 after calving compared with titers on d 0. Cows immunized with J5 bacterin had higher IgG titers (P < 0.05) on d 30 and 45 after calving and higher IgG1 titers (P < 0.05) on d 30 after calving compared with JⴢVAC immunized and control cows. Serum IgG, IgG1, and IgG2 titers of control cows, and serum IgM titers of all cows (data not shown) remained unchanged throughout the study. Whey IgG, IgG1, and IgG2 titers to E. coli J5 wholecell antigens are shown in Figures 5, 6, and 7, respectively. Immunization with the J5 bacterin enhanced (P < 0.05) the IgG1 immune response on d 14, 21, 30, and 45 after calving, and enhanced the IgG2 immune response on d 21 and 45 after dry-off, at calving, and on d 21, 30, and 45 after calving compared with control cows. The JⴢVAC immunization elevated (P < 0.05) IgG1 titers at calving, and on d 14, 21, and 45 after calving, and an increase (P < 0.05) in IgG2 titers was also observed at calving compared with control cows. Whey IgM titers between control and vaccinated cows were not different (P > 0.05; data not shown).
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Figure 4. Least-squares means for log2 serum IgG2 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with the J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Asterisks indicate a difference (P < 0.05) within treatment groups compared with values on the day of dry-off (D-0). Cows were immunized with the J5 bacterin on D-0, D+45, and at calving (C+0) or immunized with JⴢVAC on D0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving.
Figure 6. Least-squares means for log2 whey IgG1 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Cows were immunized with the J5 bacterin on D-0, D+45, and at calving (C+0), and immunized with JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving.
Figure 5. Least-squares means for log2 whey IgG antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with the J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Cows were immunized with the J5 bacterin on D-0, D+45, and at calving (C+0) and immunized with the JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving.
Figure 7. Least-squares means for log2 whey IgG2 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b) within stage of lactation indicate a difference (P < 0.05) among control cows (solid bars; n = 8), cows vaccinated with the J5 bacterin (Escherichia coli Bacterin J5 Strain, Pharmacia and Upjohn, Kalamazoo, MI; open bars; n = 8), and cows vaccinated with the JⴢVAC (Merial Limited, Athens, GA; striped bars; n = 8). Cows were immunized with the J5 bacterin on D-0, D+45, and at calving (C+0) or immunized with JⴢVAC on D-0 and D+45. All cows were challenged at 14 to 30 d postcalving. Lines above bars represent standard errors of the means. D = Days dry; C = days postcalving. Journal of Dairy Science Vol. 83, No. 10, 2000
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DISCUSSION Previous studies (4, 6, 8) that tested the efficacy of E. coli J5 vaccines have shown that immunization effectively reduced the incidence and severity of clinical coliform mastitis. The timing of vaccine administration has been based on the period of greatest risk of acquiring coliform mastitis, which has been shown to occur during the early dry period, the late dry period, and at calving (11). The commercially available J5 bacterin employed in this study recommends an immunization protocol that coincides with the periods of susceptibility mentioned above; e.g., at dry-off, mid dry period, and at calving. A recent study (12) with JⴢVAC showed that two immunizations (at dry-off and 45 d later) evoked an immune response, which elevated antibody titers and enhanced bacterial clearance from the mammary gland following intramammary challenge. In the present study, the J5 bacterin or JⴢVAC immunization increased serum and whey antibody titers over those of controls. These results concur with previous studies that investigated vaccines based on E. coli J5 (6, 7, 8, 12). One study (7) demonstrated that challenging one quarter with approximately 60 cfu of E. coli 727 produced a mild case of clinical coliform mastitis. In another study (12), two quarters of each cow were challenged with approximately 60 cfu of E. coli 727, which resulted in moderate to very severe clinical mastitis despite immunization. Cows in the current study were challenged in a single quarter to evaluate the efficacy of immunization on a more moderate infection. However, infusion of approximately 64 cfu of E. coli 727 into one quarter also induced moderate to severe clinical mastitis. The severity of infection observed in the current study may have overwhelmed the immune system of vaccinated cows, and, therefore, masked the possible effects of immunization on clinical symptoms. This phenomenon was also noted in a previous study (12). This occurred despite the reduction in colony-forming units observed in vaccinated cows. A reduction in the severity and incidence of clinical coliform mastitis has been attributed to enhanced bacterial clearance from challenged quarters and neutralization of LPS in association with elevated antibody titers to LPS core antigens (7, 8, 13). This association was observed in this study, in which immunized cows had a significantly higher rate of bacterial clearance at 144 h postchallenge than did control cows; this coincided with enhanced serum and whey IgG titers at 30 and 45 d postcalving. However, immunization with the J5 bacterin or JⴢVAC did not influence severity of clinical symptoms following intramammary challenge. This observation was noted despite the elevated presence of Journal of Dairy Science Vol. 83, No. 10, 2000
IgG1 and IgG2, which serve as opsonins of phagocytosis (5) and to neutralize endotoxin (16), respectively. A significant increase in whey IgG2 was observed at 21, 30, and 45 d postcalving in cows immunized with J5 bacterin; however, this enhanced level had no apparent influence on the effect of endotoxemia. In the present study, a marked increase in serum IgG, IgG1, and IgG2 titers was observed following the primary immunization at drying off. An immune response to the secondary immunization was evident at calving, and IgG titers remained elevated for the duration of the study. A tertiary immunization at calving with the J5 bacterin did not significantly increase antibody titers above control or JⴢVAC immunized cows until 30 to 45 d later, a period following intramammary challenge. Immunization with J5 bacterin or JⴢVAC also elevated whey IgG antibody titers to E. coli J5 whole-cell antigens. Whey IgG2 titers during the dry period (d 21 and 45) and lactation (d 21, 30, and 45 postcalving) were significantly enhanced by J5 bacterin immunization, while JⴢVAC immunized cows had elevated IgG1 titers at calving. This observation is in agreement with previously published results (12). Cows with preexisting elevated antibody titers to E. coli J5 have a reduced risk of developing clinical coliform mastitis (13). Therefore, results from this study suggest that immunization with J5 bacterin or JⴢVAC might have reduced the risk of acquiring coliform mastitis during the dry period. In conclusion, the current study suggests that immunization with the JⴢVAC (two doses) or J5 bacterin (three doses) elicits a similar immune response to E. coli J5 whole cell antigens. Cows receiving the third vaccination of J5 bacterin exhibited higher antibody titers following intramammary challenge, although the benefit of the tertiary immunization with respect to clinical symptoms and bacterial clearance from the mammary gland was not evident in this study. Severity of coliform mastitis was not influenced by either vaccine. However, immunization with the JⴢVAC or J5 bacterin enhanced bacterial clearance from challenged quarters and elevated antibody titers to E. coli J5. REFERENCES 1 Bauman, H., and J. Gauldie. 1994. The acute phase response. Immunol. Today 15:74–80. 2 DeGraves, F. J., and J. Fetrow. 1991. Partial budget analysis of vaccinating dairy cattle against coliform mastitis with an Escherichia coli J5 vaccine. J. Am. Vet. Med. Assoc. 199:451–455. 3 Gill, J. L., and H. D. Hafs. 1971. Analysis of repeated measurement of animals. J. Anim. Sci. 33:331–336. 4 Gonzalez, R. N., J. S. Cullor, D. E. Jasper, T. B. Farver, R. B. Bushnell, and M. N. Oliver. 1989. Prevention of clinical coliform mastitis in dairy cows by a mutant Escherichia coli vaccine. Can. J. Vet. Res. 53:301–305.
IMMUNIZATION AGAINST COLIFORM MASTITIS 5 Hill, A .W., D.J.S. Heneghan, T. R. Field, and M. R. Williams. 1983. Increase in specific opsonic activity in bovine milk following experimental Escherichia coli mastitis. Res. Vet. Sci. 35:222–227. 6 Hogan, J. S., K. L. Smith, D. A. Todhunter, and P. S. Schoenberger. 1992. Field trial to determine efficacy of an Escherichia coli J5 mastitis vaccine. J. Dairy Sci. 75:78–84. 7 Hogan, J. S., W. P. Weiss, K. L. Smith, D. A. Todhunter, P. S. Schoenberger, and L. M. Sordillo. 1995. Effect of an Escherichia coli J5 vaccine on mild clinical coliform mastitis. J. Dairy Sci. 78:285–290. 8 Hogan, J. S., W. P. Weiss, D. A. Todhunter, K. L. Smith, and P. S. Schoenberger. 1992. Efficacy of an Escherichia coli J5 mastitis vaccine in an experimental challenge trial. J. Dairy Sci. 75:415–422. 9 National Mastitis Council. 1987. Current Concepts of Bovine Mastitis. 3rd ed. Natl. Mastitis Counc., Inc., Arlington, VA. 10 SAS/STAT User’s Guide, Release 6.03. 1988. SAS Inst. Inc., Cary, NC.
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11 Smith, K. L., D. A. Todhunter, and P. S. Schoenberger. 1985. Environmental mastitis: cause, prevalence, prevention. J. Dairy Sci. 68:1531–1553. 12 Tomita, G. M., S. C. Nickerson, W. E. Owens, and B. Wren. 1998. Influence of route of vaccine administration against experimental intramammary infection caused by Escherichia coli. J. Dairy Sci. 81:2159–2164. 13 Tyler, J. W., J. S. Cullor, B. I. Osburn, R. B. Bushnell, and B. W. Fenwick. 1988. Relationship between serologic recognition of Escherichia coli O111:B4 (J5) and clinical coliform mastitis in cattle. Am. J. Vet. Res. 49:1950–1954. 14 Tyler, J., H. Spears, J. Cullor, and W. Smith. 1991. Antigenic homology among Gram-negative organisms isolated from cattle with clinical mastitis. J. Dairy Sci. 74:1235–1242. 15 Tyler, J. W., H. Spears, and R. Nelson. 1992. Antigenic homology of endotoxin with a coliform mastitis vaccine strain, Escherichia coli O111:B4 (J5). J. Dairy Sci. 75:1821–1825. 16 Zeigler, E. J., H. Douglas, J. E. Sherman, C. E. Davis, and A. I. Braude. 1973. Treatment of E. coli and Klebsiella bacteremia in agranulocytic animals with antiserum to a udp-gal epimerasedeficient mutant. J. Immunol. 111:433–439.
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