Influence of Route of Vaccine Administration Against Experimental Intramammary Infection Caused by Escherichia coli1

Influence of Route of Vaccine Administration Against Experimental Intramammary Infection Caused by Escherichia coli 1 G. M. TOMITA,* S. C. NICKERSON,* W. E. OWENS,* and B. WREN† *Hill Farm Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Route 1, Box 10, Homer 71040 †Merial Limited, 115 Transtech Drive, Athens, GA 30601

ABSTRACT

INTRODUCTION

The route of immunization of a commercially available Escherichia coli J5 bacterin was investigated. Jersey cows were randomly assigned to one of three treatment groups: 1 ) unvaccinated (control), 2 ) vaccinated subcutaneously in the neck, and 3 ) vaccinated in the area of the supramammary lymph node. Cows were vaccinated at drying off and at 2 wk prior to anticipated calving. Two quarters of each cow were challenged with approximately 60 cfu of E. coli at 14 d postcalving. Route of immunization in the neck or the area of the supramammary lymph node did not influence severity of coliform mastitis. However, the mean number of colony-forming units of E. coli recovered from challenged quarters was significantly lower for vaccinated cows than for control cows at 24 h postchallenge. A quicker milk yield recovery following intramammary challenge was also observed for vaccinated cows. Serum immunoglobulin ( I g ) G, IgG1, and IgG2 and whey IgG1 and IgG2 antibody titers against E. coli J5 whole-cell antigens were significantly enhanced in vaccinated cows. Somatic cell counts in milk from challenged quarters and rectal temperatures following intramammary challenge were not different for cows across treatment groups. Immunization did not prevent intramammary infection. ( Key words: immunization, supramammary lymph node, mastitis, Escherichia coli J5)

Herd health management practices, such as teat dipping and dry cow antibiotic therapy, are not effective in the control of coliform mastitis ( 1 1 ) primarily because of continuous exposure of teat ends to coliform pathogens. Control can be achieved by the elimination of coliform pathogens from the environment of cows, but this approach is not economically feasible. An alternative strategy would be to enhance the resistance of the cow against coliform IMI through active immunization. Lipopolysaccharide ( LPS) , which is present on the outer cell wall of coliform bacteria, is responsible for the acute clinical symptoms associated with coliform mastitis ( 1 ) . The core and lipid A regions of LPS possess antigenic homology among coliform pathogens and, as such, have been targeted as vaccine antigens. A mutant strain of Escherichia coli O111:B4 ( J 5 ) has a unique characteristic: the core and lipid A antigens of LPS are exposed, and immunization with E. coli J5 produces antibodies that are crossreactive with other coliform pathogens (13, 14). Studies have shown that dairy cows with elevated, naturally occurring antibody titers to E. coli J5 have a decreased risk of developing clinical coliform mastitis ( 1 2 ) and that immunization with an E. coli J5 bacterin enhances antibody titers to E. coli J5 in serum and mammary secretions (3, 5, 6, 7). Cows that were immunized with the bacterin also had reduced severity and lower rates of clinical coliform mastitis than did unimmunized controls (3, 5, 6, 7). Results from a preliminary study utilizing a commercially available E. coli J5 bacterin (J·VAC; Merial Limited, Athens, GA) suggested that a greater immune response to E. coli J5 could be achieved by vaccination in the area of the supramammary lymph node ( SMLN) as opposed to subcutaneous injection in the neck. However, the protective effect of immunization with J·VAC against coliform mastitis was not

Abbreviation key: LPS = lipopolysaccharide, SMLN = supramammary lymph node.

Received December 22, 1997. Accepted March 23, 1998. 1Approved for publication by the director of the Louisiana State University Agricultural Experiment Station as Manuscript Number 97-80-0465. 1998 J Dairy Sci 81:2159–2164

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determined. The present study was conducted to determine the effect of route of immunization on the efficacy of J·VAC against clinical responses to intramammary bacterial challenge and on humoral immunity. MATERIALS AND METHODS Immunization Jersey cows with no prior history of coliform mastitis were randomly assigned according to age and parity to one of three treatment groups: 1 ) unvaccinated (control; n = 8), 2 ) vaccinated subcutaneously in the neck ( n = 6), and 3 ) vaccinated in area of the SMLN ( n = 8). Cows were immunized with J·VAC at drying off ( d 0 ) and received a secondary vaccination 2 wk prior to anticipated calving ( d 45) as recommended by the manufacturer. Rectal temperature and general health of vaccinated cows were monitored prior to and 12, 24, 48, and 72 h following each vaccination. Intramammary Challenge Two uninfected quarters of each cow were infused 5 to 6 h after the morning milking with approximately 60 cfu of E. coli suspended in 1 ml of sterile PBS (pH 7.2). Quarters were challenged 14 d postcalving. The challenge strain was previously isolated from a clinical mastitic quarter ( 6 ) . Aseptic milk samples from challenged quarters were collected ( 8 ) immediately prior to challenge and at 4, 8, 12, 16, 20, 24, 48, 72, 96, 120, and 144 h postchallenge. The number of colony-forming units per challenge dose and colonyforming units per milk sample were determined by MacConkey agar pour plates (Becton Dickinson and Co., Cockeysville, MD), and SCC were determined using a Fossomatic cell counter (A/N Foss Electric, Hillerød, Denmark). Rectal temperatures of cows were also monitored at the time intervals mentioned previously. Milk yield was monitored at 12-h intervals beginning 12 h prior to challenge through 132 h postchallenge. All cows were milked twice daily. Antibody Titers Serum and whey samples were collected on d 0, 21, and 45, at calving, and on d 14, 21, 30, and 45 after calving and were subjected to ELISA ( 1 3 ) to determine antibody titers to E. coli J5 whole-cell antigens. The following isotypes were assayed from those samples: IgG, IgG1, IgG2, and IgM. Journal of Dairy Science Vol. 81, No. 8, 1998

Statistical Analysis Comparisons of colony-forming units, SCC, rectal temperatures, milk yields, and antibody titers among treatment groups were analyzed using the mixed models procedure for measures repeated across time ( 2 ) . Colony-forming units, SCC, and milk yield 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 ( 1 0 ) and the following model: Yijk = m + Ti + Cj( T i) + Sk + STki + e´ijk where Yijk = observed colony-forming units, SCC, rectal temperature, milk yield, or antibody titer; m = effect common to observations; Ti = fixed effect of treatment i; Cj( T i) = random effect associated with cow j in treatment i; Sk = fixed effect of sample time k; STki = fixed effect associated with treatment i and sample time k; and e´ijk = random error. RESULTS Immunization with J·VAC did not cause any adverse reactions at either injection site. Minimal swelling of the injection site (<2.5 cm), which disappeared within 48 h, was observed for cows subcutaneously injected in the neck. No adverse reactions were noted for cows immunized in the area of the SMLN. Febrile response to immunization was not evident in vaccinated cows. Colony-forming units of E. coli and SCC recovered from challenged quarters, rectal temperatures, milk yields, and antibody titers were not influenced by route of immunization. Therefore, data from vaccinated cows were combined. The challenge dose of E. coli and colony-forming units per milliliter of milk recovered from challenged quarters are shown in Figure 1. The number of bacteria infused into each quarter of control cows (65.2 ± 2.1 cfu) and vaccinated cows (62.9 ± 3.1 cfu) was not different ( P > 0.05). The number of colony-forming units of E. coli recovered from challenged quarters of all cows peaked at 12 h postchallenge and began to decrease in both treatment groups by 16 h. Compared with control cows, vaccinated cows began to exhibit a reduction ( P < 0.05) in the number of colony-forming units by 24 h postchallenge. This association continued through 144 h postchallenge.

ROUTE OF IMMUNIZATION

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) between control cows ( ◊; n = 16) and cows immunized with J·VAC (Merial Limited, Athens, GA; ⁄; n = 28). Bars represent standard errors of the means. CD = Challenge dose.

Rectal temperatures and SCC between control and vaccinated cows were not different ( P > 0.05; data not shown). A typical response to induced coliform mastitis was observed. Rectal temperatures began to increase 8 h following intramammary challenge and peaked at 16 h. Temperatures returned to prechallenge values by 24 h. Milk SCC peaked by 12

Figure 2. Least squares means for log10 milk yield (kilograms × 10) of control cows ( ⁄; n = 8 ) and cows immunized with J·VAC (Merial Limited, Athens, GA; ♦; n = 14). Cows were challenged at 0 h. Different letters (a, b ) within hours indicate a difference ( P < 0.05) between control and vaccinated cows. Asterisks indicate a difference ( P < 0.05) between prechallenge and postchallenge milk yields. Bars represent standard errors of the means.

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Figure 3. Least squares means for log2 serum IgG antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b ) within stage of lactation indicate a difference ( P < 0.05) between control cows ( ◊; n = 8 ) and cows immunized with J·VAC (Merial Limited, Athens, GA; ⁄; n = 14). Asterisks indicate a difference ( P < 0.05) within treatment groups compared with values on the day of dry-off ( D – 0). Cows were immunized at dryoff and on d 45 of the dry period and were challenged at 14 d postcalving. Bars represent standard errors of the means. D = Days dry; C = days postcalving.

h postchallenge and remained elevated (>10 6/ml) throughout the study. Milk yield results are shown in Figure 2. A decrease ( P < 0.05) in the milk yield of control cows began 48 h after challenge and was most prominent at 60 h postchallenge compared with the prechallenge milk yield. Recovery to the prechallenge milk yield was not evident until 72 h. A decline ( P < 0.05) in the milk yield of vaccinated cows compared with the prechallenge yield was observed 36 h after challenge; milk yield recovered to the prechallenge value 12 h later. Differences ( P < 0.05) between treatment groups were observed at 36 and 60 h. Similar trends in serum IgG (Figure 3 ) and IgG1 (Figure 4 ) antibody titers to E. coli J5 whole-cell antigens were observed. Vaccinated cows had higher ( P < 0.05) antibody titers on d 21, at calving, and 14, 30, and 45 d after calving compared with control cows. Antibody titers of vaccinated cows were also elevated ( P < 0.05) on d 21 and d 21, 30, and 45 after calving compared with titers on d 0. Serum IgG2 antibody titers of vaccinated cows were higher ( P < 0.05) on d 45, at calving, and on d 14 after calving compared with the serum IgG2 antibody titers of control cows (Figure 5). Immunization also enhanced IgG2 titers ( P < 0.05) at 3 wk following the primary vaccination ( d 21), at 2 wk after the secondary vacciJournal of Dairy Science Vol. 81, No. 8, 1998

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Figure 4. Least squares means for log2 serum IgG1 antibody titers to Escherichia coli J5 whole-cell antigens. Different letters (a, b ) within stage of lactation indicate a difference ( P < 0.05) between control cows ( ◊; n = 8 ) and cows immunized with J·VAC (Merial Limited, Athens, GA; ⁄; n = 14). Asterisks indicate a difference ( P < 0.05) within treatment groups compared with values on the day of dry-off ( D – 0). Cows were immunized at dryoff and on d 45 of the dry period and were challenged at 14 d postcalving. Bars represent standard errors of the means. D = Days dry; C = days postcalving.

nation (calving), on the day of challenge (14 d after calving), and throughout the remainder of the study compared with preimmunization titers on d 0. 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 IgG1 and IgG2 titers to E. coli J5 whole-cell antigens are shown in Figures 6 and 7, respectively. Vaccinated cows had higher ( P < 0.05) IgG1 titers on d 21 and 45, at calving, and on d 14, 30, and 45 after calving. Immunoglobulin G2 immune response was also enhanced ( P < 0.05) on d 21 and 45 and at calving compared with control cows. Vaccinated cows also had elevated ( P < 0.05) IgG1 and IgG2 titers during the dry period ( d 21 and 45), at calving, and during early lactation (30 and 45 d after calving) compared with preimmunization titers on d 0. Whey IgG and IgM titers between control and vaccinated cows were not different ( P > 0.05; data not shown). DISCUSSION A previous study utilizing a Staphylococcus aureus bacterin ( 9 ) demonstrated that immunization in the area of the SMLN versus intramuscular immunization enhanced local immune response in the mammary gland. Immunization via the SMLN led to an increase in the number of cells in mammary tissue Journal of Dairy Science Vol. 81, No. 8, 1998

that produce antibody. Similarly, a pilot study employing J·VAC suggested that immunization in the area of the SMLN initiated a greater immune response than did subcutaneous or intramuscular immunization (S. C. Nickerson, 1995, unpublished data). This phenomenon was not apparent in the present study. Antibody titers of cows vaccinated in the neck or in the SMLN were similar in magnitude and duration. However, immunization with J·VAC did increase serum and whey antibody titers over those of controls. These results concur with previous studies that utilized an E. coli J5 bacterin (5, 6, 7). A recent study ( 6 ) demonstrated that challenging one quarter with approximately 60 cfu of E. coli produced a mild case of clinical coliform mastitis. In the present study, 2 quarters of each cow were challenged with approximately 60 cfu of E. coli, which resulted in moderate to severe clinical mastitis in all treatment groups. The initial replication of E. coli following intramammary infusion into 2 quarters and the subsequent release of LPS might have temporally caused the cows to be unable to ameliorate the infection and endotoxemia and, therefore, masked the possible effects of immunization with J·VAC on clinical symptoms. This result occurred despite the reduction in colony-forming units observed for vaccinated cows. Data suggest that immunization may not reduce clini-

Figure 5. 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) between control cows ( ◊; n = 8 ) and cows immunized with J·VAC (Merial Limited, Athens, GA; ⁄; n = 14). Asterisks indicate a difference ( P < 0.05) within treatment groups compared with values on the day of dry-off ( D – 0). Cows were immunized at dryoff and on d 45 of the dry period and were challenged at 14 d postcalving. Bars represent standard errors of the means. D = Days dry; C = days postcalving.

ROUTE OF IMMUNIZATION

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) between control cows ( ◊; n = 8 ) and cows immunized with J·VAC (Merial Limited, Athens, GA; ⁄; n = 14). Asterisks indicate a difference ( P < 0.05) within treatment groups compared with values on the day of dry-off ( D – 0). Cows were immunized at dryoff and on d 45 of the dry period and were challenged at 14 d postcalving. Bars represent standard errors of the means. D = Days dry; C = days postcalving.

cal symptoms of coliform mastitis when multiple quarters are infected. However, an enhanced immune response to core LPS antigens may be beneficial because of the quicker milk yield recovery following acute coliform mastitis. A reduction in the severity of coliform mastitis has been attributed to enhanced bacterial and LPS clearance from challenged quarters in association with elevated antibody titers to E. coli J5 (6, 7). The same association was observed in this study; vaccinated cows had a significantly higher rate of bacterial clearance than did control cows in conjunction with enhanced IgG2 titers, which are proposed to be opsonins of pathogens for phagocytosis ( 4 ) . But, immunization did not influence severity of clinical symptoms. This observation was noted despite the elevated presence of IgG1, which is proposed to neutralize endotoxin associated with Gram-negative bacterial infections (15). In the present study, a marked increase in serum IgG, IgG1, and IgG2 titers was observed on d 21 following the primary immunization at drying off and declined to preimmunization concentrations by the time cows received their secondary immunization at d 45. An immune response to the secondary immunization was evident at calving, and IgG titers remained elevated for the duration of the study. Immunization with J·VAC also elevated whey IgG1 and IgG2 anti-

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body titers to E. coli J5 whole-cell antigens during the dry period. This observation has not been previously published. Tyler et al. ( 1 2 ) reported that cows with elevated antibody titers to E. coli J5 have a reduced risk of developing clinical coliform IMI during lactation. Therefore, results from this study suggest that vaccinated cows may also be at a reduced risk of acquiring coliform IMI during the dry period because of the increased presence of antibodies that recognize core LPS epitopes of E. coli J5. An increase in whey IgG in association with enhanced IgG1 and IgG2 titers was not evident because of variations in IgG end point titers between treatment groups. A slight increase in serum IgG of control cows was noted following intramammary challenge, but the increased serum IgG was not significantly higher than the prechallenge value. In conclusion, results from the current study suggest that immunization in the area of the SMLN is not advantageous compared with immunization in the neck. Severity of coliform mastitis induced experimentally was not influenced by route of immunization. However, immunization with J·VAC enhanced bacterial clearance from challenged quarters and elevated antibody titers to E. coli J5, which might have influenced the more rapid milk yield recovery observed for vaccinated cows compared with control cows.

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) between control cows ( ◊; n = 8 ) and cows immunized with J·VAC (Merial Limited, Athens, GA; ⁄; n = 14). Asterisks indicate a difference ( P < 0.05) within treatment groups compared with values on the day of dry-off ( D – 0). Cows were immunized at dryoff and on d 45 of the dry period and were challenged at 14 d postcalving. Bars represent standard errors of the means. D = Days dry; C = days postcalving. Journal of Dairy Science Vol. 81, No. 8, 1998

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REFERENCES 1 Bauman, H., and J. Gauldie. 1994. The acute phase response. Immunol. Today 15:74–80. 2 Gill, J. L., and H. D. Hafs. 1971. Analysis of repeated measurement of animals. J. Anim. Sci. 33:331–336. 3 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. 4 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. 5 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. 6 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. 7 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.

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8 National Mastitis Council. 1987. Current Concepts of Bovine Mastitis. 3rd ed. Natl. Mastitis Counc., Inc., Arlington, VA. 9 Nickerson, S. C., W. E. Owens, and R. L. Boddie. 1993. Effect of a Staphylococcus aureus bacterin on serum antibody, new infection, and mammary histology in nonlactating dairy cows. J. Dairy Sci. 76:1290–1297. 10 SAS/STAT User’s Guide, Release 6.03. 1988. SAS Inst., Inc., Cary, NC. 11 Smith, K. L., D. A. Todhunter, and P. S. Schoenberger. 1985. Environmental mastitis: cause, prevalence, prevention. J. Dairy Sci. 68:1531–1553. 12 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 ( J 5 ) and clinical coliform mastitis in cattle. Am. J. Vet. Res. 49:1950–1954. 13 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. 14 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. 15 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.