Field Trial to Detennine Efficacy of an Escherichia coli J5 Mastitis Vaccine 1 J. S. HOGAN, K. L. SMITH, D. A. TODHUNTER, and P. S. SCHOENBERGER Department of Dairy Science
The Ohio State University Ohio Agricultural Research and Development Genter Wooster 44691
ABSTRACT
New IMI caused by coliforms are highly associated with the 2-wk period prior to calving and early lactation (15). The high rate of coliform IMI at calving coincides with a period of reported immunosuppression (3, 10). An effective vaccine that would increase resistance to coliform infections during these periods would reduce significantly the losses caused by this disease. Considerable attention has been given to the protective effects of vaccinating laboratory animals and humans with the Rc rough mutant Escherichia coli (0111:B4) 15. Escherichia coli 15 lipopolysaccharide (LPS) lacks 0polysaccharide chains, thereby exposing the core antigens of LPS (4). The core antigens of LPS appear to be highly conserved among Gram-negative bacteria compared with the extremely heterogenous O-polysaccharide antigens. Both passive and active immunization with E. coli 15 protected laboratory animals against endotoxic shock and bacteremia by heterologous strains (2, 12, 17). Vaccinating dairy cows with E. coli 15 bacterin during the dry period and early lactation significantly reduced the incidence of clinical mastitis during the first 90 d of lactation (5). However, the investigators did not determine whether immunization reduced the incidence of IMI. The purpose of the current trial was to determine effects of an E. coli 15 bacterin on reducing naturally occurring IMI and clinical mastitis in a commercial dairy herd.
Eflficacy of an Escherichia coli (0111:B4) 15 bacterin for preventing naturally occurring IMI and clinical mastitis was tested in a 2.5-yr field trial in a 225~ow commercial herd. Cows with odd-numbered identification were vaccinated, and cows with evennumbered identification served as unvaccinated controls for each lactation during the study. Immunizations were subcutaneous on the upper part of the rib cage just posterior to the scapula at drying off, 30 d after drying off, and at calving. Percentage of quarters infected at calving with Gram-negative bacteria did not differ between treatment groups. A total of 67% of Gram-negative bacterial IMI present at calving in control cows became clinical during the first 90 d of lactation compared with 20% in vaccinated cows. Rate of Gram-negative bacterial clinical mastitis was higher in control cows than in vaccinated cows during the first 90 d of lactation. Immunization with the E. coli 15 bacterin did not reduce level of Gram-negative bacterial IMI at calving but did reduce incidence of clinical mastitis. (Key words: Escherichia coli, vaccine, mastitis) Abbreviation key: LPS
= lipopolysaccharide.
INTRODUCTION
MATERIALS AND METHODS
Gram-negative bacterial IMI are common causes of toxic clinical mastitis in dairy cattle.
Cooperating Herd
Received June 12, 1991. Accepted September 6, 1991. 1Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 99-91.
Experimental cows were in a commercial herd of approximately 225 lactating Holsteins. Lactating cows were in total confinement freestall housing. Free stalls were bedded with washed sand Cows were milked twice daily in a double-eight herringbone parlor. Milking hygiene included premilking and postmilking dis-
1992 J Dairy Sci 75:78-84
78
ESCHERICHIA COli 15 FIEID TRIAL
infection of teats with 5.25% sodium hypochlorite. All cows were dried off by abrupt cessation of milking, and all quarters were drytreated with an FDA approved dry cow therapy product. Dry cows were pastured with constant access to loose housing bedded with long straw. Two weeks prior to anticipated calving, cows were transferred to a 4.6- x 9.2-m maternity area and remained there until the fIrst regularly scheduled milking after parturition. The study was initiated in March 1988 and concluded in October 1990. Rolling herd averages were 8235 kg of milk and 307 kg of fat at the beginning of the study and 9600 kg of milk and 330 kg of fat at termination of the experiment. Rolling herd average SCC was 164,0001 00 of milk in March 1988 and 109,000/00 of milk in October 1990. Experimental Design
Cows entered the trial the day of drying off. Cows with odd-numbered identification were vaccinated, and cows with even-numbered identification served as unvaccinated controls for each lactation during the study. Escherichia coli bacterin was 5 00 of 109 cells of boiled E. coli J5/ml plus 1 00 of Freund's incomplete adjuvant (Difco Laboratories, Detroit, MI). Immunizations were at drying off, 30 dafter drying off, and within 24 h after parturition. Immunizations were subcutaneous on the upper part of the rib cage just posterior to the scapula. Milk samples
Bacteriological status of mammary quarters was determined by analyses of samples collected utilizing the following scheme. Duplicate quarter foremilk samples were collected aseptically by the herd manager from 1) all cows once during the 7- .05). between vaccinated cows and unvaccinated Rate of total clinical cases was .583/1000 controls (P > .05). Level of Gram-negative cow-days in parity 3 control cows and .095/ bacterial IMI was greater (P < .05) in control 1000 cow-days in parity 3 vaccinated cows cows than in vaccinated cows during yr 1 of (Table 3; P < .05). Gram-negative bacterial the study (2.1 and .4% quarters, respectively). Gram-negative bacterial IMI at calving in- clinical mastitis rate in parity >3 control cows creased during yr 2 to 2.3% of quarters com- was fourfold greater (P < .05) than the rate for pared with .4% during yr 1 in vaccinated cows. parity >3 vaccinated cows. Within lactation 1 Percentage of quarters infected with Gram- (Table 4), control cows had a greater (P < .05) negative bacteria at calving in unvaccinated rate of clinical Gram-negative bacterial masticows did not differ among the years. Percent- tis (.300/1000 cow-days) than vaccinated cows age of quarters infected with Gram-negative (.065/1000 cow-days). Rates of clinical Grambacteria at calving did not differ among parity negative bacterial mastitis did not differ begroups or between lactation 1 and 2 cows. tween lactation 1 and lactation 2 cows within Percentage distribution of species within experimental treatment groups. Both rates of total clinical cases and clinical Gram-negative bacterial IMI at calving in control cows was E. coli, 33.3%, Serratia species, Gram-negative cases were greater in control 33.3%; Proteus species, 25%; and Klebsiella cows than in vaccinated cows during yr 1 of species, 8.3%. Percentage distribution of spe- the study (Table 5). Rates of clinical mastitis cies within Gram-negative bacterial IMI at did not differ between treatment groups during calving in vaccinated cows was E. coli, 50%; yr 2 and 3. Rate of clinical Gram-negative Serratia species, 20%; Pseudomonas species, mastitis cases was greatest in yr 1 for control 20%; and Klebsiella species, 10%. A total of cows, but rates did not differ among years of 66.7% of Gram-negative bacterial IMI at calv- the study in vaccinated cows. In summary, 7 of (lactation 1 first lactation on the trial; lactation 2 = second and third lactations on the trial).
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ESCHERICHIA COll 15 FIELD TRIAL
TABLE 2. Rate of clinical mastitis! in Escherichia coli 15 vaccinated (15) and unvaccinated cows (C) during a 2.5-yr field trial in a commercial dairy. First 90 d3 Bacteriological status
C
15
Gram-negative bacilli Environmental streptococci Staphylococcus species Actinomyces pyogenes Bacteriologically negative Total
.2558 .064 .021 .064 .106 .511
.047b .5708 .094.000 .023 .063 .023 .127 .141 .127 .328 .887
15
C
.140b
210 .070 .000 .351 .772
"~ates with differing superscripts within rows and comparisons differ (P < .05). 1Rate of clinical mastitis = number of clinical cases per 1000 cow-days. 2Rate per total lactation. 3Rate per f1I'St 90 d of lactation.
the 12 (58%) Gram~negative bacterial clinical cases in unvaccinated cows occurred during the first 90 d of lactation in parity >3 cows during the 1st yr of the study compared with 0 of 2 cases in vaccinated cows. Percentage distribution of species within Gram-negative bacterial clinical mastitis cases in control cows was E. coli, 50%; Serratia species, 25%; Klebsiella species, 16.7%; and Proteus species, 8.3%. Both Gram-negative bacterial clinical cases in vaccinated cows were E. coli. DISCUSSION
Vaccinating dairy cows with an E. coli J5 bacterin during the dry period and early lactation significantly reduced rate of Gramnegative bacterial clinical mastitis during this 2.5-yr study. These results agree with those of Gonzales et al. (5) in which incidence of clini-
cal mastitis was reduced by immunizing cows with the same vaccine during a 1.5-yr study in two California dairy herds. In addition, the magnitude of reduction was similar between trials. Rate of Gram-negative bacterial clinical mastitis was 4.1-fold lower in vaccinated cows versus controls in the present study compared with a 4.9-fold difference in the California trial. Data in the present trial suggest that protection offered by the vaccine was greatest for parity >3 cows in early lactation. TIlls corresponds with results of previous surveys showing that older cows during the first weeks following calving is the population at greatest risk to Gram-negative bacterial clinical mastitis (15). However, the data suggest that positive effects of vaccination were not restricted to older cows. Although differences were not significant, rates of Gram~negative bacterial clinical mastitis also tended to be higher in
TABLE 3. Rate of clinical mastitis! in Escherichia coli 15 vaccinated (15) and unvaccinated cows (C) during a 2.5-yr field trial in a commercial dairy by parity groups. Parity 2 Bacteriological status Gram-negative bacilli Environmental streptococci Staphylococcus species Actinomyces pyogenes Bacteriologically negative Total
C
.009 .198 .000 .000 .000 .280
Parity 3 15
Parity >3 15
C
.000 .194 .116 .097 .000.000 .000.000 .231 .291 .347 .5838
.000 .095 .000 .000 .000 .095 b
15
C 8
.388 .000 .038 .113 .075 .564
.085b .085 .043 .043 .171 .427
a,~tes with differing superscripts within rows and comparisons differ (P < .05). IRate of clinical mastitis = number of clinical cases per 1000 cow-days. lournal of Dairy Science Vol. 75, No.1, 1992
82
HOGAN ET AL.
TABLE 4. Rate of clinical mastitis l in Escherichia coli 15 vaccinated (15) and unvaccinated cows (C) during a 2.5-yr field ttial in a commercial dairy by number of laerations a cow was on the ttial. Lactation Bacteriological status Gnan-negative bacilli Environmental streptococci Staphylococcus species Actinomyces pyogenes Bacteriologically negative Total
Lactation
C
IS
.300"
.065 b
C
15
.155 .13O.()(l() .030.000 .032 .155 .129 .077 .389 .386
.099 .032 .030 .120 .570
.000 .000 .000 .000 .175 .175
a,~tes with differing superscripts within rows and comparisons differ (P < .05). IRate of clinical mastitis
= number of clinical
cases per 1000 cow-days.
2Rate among cows completing only one dry period on the ttial. 3Rate among cows completing second and third dry periods on the trial.
controls than in vaccinates in the other parity groups. Cows that underwent a second and third regimen of vaccination did not appear to have enhanced protection compared with those completing only one series of inununizations. This was evidenced by the fact that rates of clinical mastitis did not differ between lactation 1 and 2 vaccinated cows. Conclusions drawn from this study concerning the value of vaccinating cows in ensuing dry periods and lactations should be qualified by accounting for the relatively small number of animals completing more than one dry period. A total of 44 and 4 vaccinated cows completed two and three immunization regimens, respectively. A comparison of Gram-negative bacterial clinical cases within these 48 cows showed that one clinical
case occurred in cows during the first lactation on the trial compared with zero during second and subsequent lactations. Differences in rates of clinical mastitis between treatment groups changed during the trial. Vaccinated cows had a lower rate of Gram-negative bacterial clinical cases than controls during the first 12 mo, but differences between groups were not significant the last 18 mo. This apparent change in efficacy was not a result of decreased protection afforded by the vaccine because rates of clinical mastitis did not differ throughout the study in vaccinated cows. Rates of Gram-negative clinical mastitis in control cows were significantly reduced the last 18 mo of the study compared with the first 12 mo. The decrease in clinical mastitis was possibly caused by positive changes in one or
TABLE 5. Rate of clinical mastitis l in Escherichia coli 15 vaccinated (15) and unvaccinated cows (C) during a 2.5-yr field ttial in a commercial dairy by year of study. y~
1
2
3
Bacteriological status
C
15
C
IS
C
15
Gnan-negative bacilli Environmental streptococci Staphylococcus species Actinomyces pyogenes Bacteriologically negative Total
.430"' .000 .048 .048 .096 .621 a
.000b .000 .051 .000 .101 .152b
.124 .124 .000 .000 .083 .330
.095 .189 .000 .047 .047 .378
.000 .000 .000 1.104 .552 1.657
.000 .000 .000 .000 1.739 1.739
a,~tes with differing S1Iperscripts within rows and comparisons differ (P < .05). IRate of clinical mastitis number of clinical cases per 1000 cow-days.
=
2yr I = April
1988 to March 1989; yr 2 = April 1989 to March 1990; and yr 3 = April 1990 to September 1990.
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ESCHERICHIA COll 15 FIELD TRIAL
more management practices in the herd. Although care was taken by both herd managers and researchers conducting the trial to ensure that management practices other than experimental treatments did not differ between treatment groups, changes in husbandry practices were left to the discretion of the herd managers. These data illustrate the advantage of longer field trials to document the efficacy of vaccines as the incidence of naturally occurring infections and clinical cases changes within a herd. The advantages afforded by immunizing with the E. coli J5 bacterin will be minimized as environmental factors either reduce the exposure of cows to Gram-negative bacterial pathogens or enhance nonspecific host defense mechanisms. Immunization of cows with E. coli J5 during the dry period did not protect cows from IMI at parturition. Vaccination reduced the percentage of quarters infected at calving with Gram-negative bacteria the 1st yr but did not affect prevalence for the remainder of the study. Control and vaccinated cows also had similar percentage distributions of Gramnegative bacterial species within IMI diagnosed at calving. The most striking difference between treatment groups was that 66.7% of IMI in controls became clinical during early lactation compared with 20% in vaccinates. These data imply that the E. coli J5 vaccine did not prevent the occurrence of IMI but did reduce the severity of the disease. The mechanism of action by which immunization with E. coli J5 provides protection appears to be related to increased antibody to the highly conserved LPS core antigens. Crossreactivity of antisera from vaccinated animals to heterologous smooth Gram-negative bacteria was due to antibody directed against core antigens (1, 13, 14). The protection afforded by vaccinating with E. coli J5 has been hypothesized as being due to increased opsonization of heterologous bacteria and detoxification of LPS by antibody (2, 17). Although serological testing was not within the practical limitations of this study, an earlier study showed that cows vaccinated with the bacterin had increased serum and milk: titers of IgM and opsonic activity against a heterologous E. coli strain (9). The relevance of increased IgM concentration in decreasing mastitis is questionable because milk from unvaccinated cows
has sufficient opsonic actlVity (7). Experimental challenge trials revealed that vaccination did not prevent IMI, and antibody titer response to immunization was minimal prior to infection (6). However, serum IgG titer increased more in vaccinated cows than in unvaccinated controls 6 wk following experimental challenge. Mastitis data in the current and previous studies concur with experiments in laboratory animals that suggest the primary protective action of the E. coli J5 vaccine is the reduction of systemic responses from endotoxemia following Gram-negative bacterial infections, thus reducing the severity of the disease. ACKNOWLEDGMENTS
The authors thank Jim Collor, University of California-Davis, for kindly providing the antigen; the Ayers Farm, Inc. for its unselfish cooperation; and the Ohio Dairy Farmers' Federation for partial financial support of this experiment. REFERENCES 1 Baumgartner, I. D., T. X. O'Brien, T. N. Kirkland, M P. Glauser, and B. I. Ziegler. 1987. Demonstration of cross-reactive antibodies to smooth Gram-negative bacteria in antiserum to Escherichia coli 15 J. Infecl Dis. 156:136. 2 Braude, A. I., E. J. Ziegler, H. Douglas, and J. A. McCutchan. 1977. Antibody to cell wall glycolipid of Gram-negative bacteria: induction of immunity to bacteremia and endotoxemia. I. Infect. Dis. 136:S167. 3 Craven, N., and M. R. Williams. 1985. Defenses of the bovine mammary gland against infection and prospects for their enhancements. Vet. Immunol. Immunopathol. 2:71. 4 ElOOm, A. D., and E. C. Heath. 1965. The biosynthesis of cell wall lipopolysaccharide in Escherichia coli. J. Biol. Chern.. 5:1919. 5 Gonzales, R. N., J. S. Cullor, D. B. 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. Vel Res. 53:301. 6 Hill, A. W. 1991. Vaccination of cows with rough Escherichia coli mutants fails to protect against experimental intramammary bacterial challenge. Vel Res. Common. 15:7. 7 Hill, A. W., DJ.S. Heneghan, and M. R. Williams. 1983. The opsonic activity of bovine milk whey for the phagocytosis and killing by neutrophils of encapsulated and non-encapsulated Escherichia coli. Vet. Microbiol. 8:293. 8 Hogan, I. S., K. L. Smitb. K. H. Hoblet, P. S. Schoenberger, D. A. Todhunter, W. D. Hueston, D. B. PritchJournal of Dairy Science Vol. 75, No.1, 1992
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ard, G. L. Bowman, L. E. Heider, B. L. Brockett, and H. R. Conrad. 1989. Field survey of mastitis in low somatic cell count herds. I. Dairy Sci. 72:1547. 9 Hogan, I. S., D. A. Todhunter, G. M. Tomita, K. L. Smith, and P. S. Schoenberger. 1992. Opsonic activity of bovine serum and mammary secretion after Escherichia coli 15 vaccination. I. Dairy Sci. 75:12. 10 Kehrli, M. E., B. I. Nonnecke, and I. A. Roth. 1989. Alterations in bovine neutrophil function during the periparturient period. Am. I. Vet. Res. 50:207. 11 Koneman, E. W., S. D. Allen, V. R. Dowell, and H. M. Sommer. 1983. The enterobacteriaceae. Page 57 in Color atlas and textbook of diagnostic microbiology. 2nd ed. I. B. Lippincott Co., New York, NY. 12 Marks, M. I., E. I. Ziegler, H. Douglas, L. B. Corbeil, and A. I. Braude. 1982. Induction of immunity against lethal Haemophilus influenzae type b infection by Escherichia coli core lipopolysaccharide. I. CIin. Invest. 69:742.
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13 McCallus, D. E., and N. L. Norcross. 1987. Antibody specific for Escherichia coli 15 cross-reacts to various degrees with an Escherichia coli clinical isolate grown for different lengths of time. Infect. Immun. 55:1042. 14 Mulharia, L. M., G. Crockford, W. C. Bogard, and R.E.W. Hancock. 1984. Monoclonal antibodies specific for Escherichia coli 15 lipopolysaccharide: crossreaction with other Gram-negative bacterial species. Infect. Immun. 45:631. 15 Smith, K. L., D. A. Todhunter, and P. S. Schoenberger. 1985. Environmental mastitis: cause, prevalence, prevention. I. Dairy Sci. 68:1531. 16 Sokal, R. R., and F. I. Rohlf. 1981. Biometry. 2nd ed. W. H. Freeman and Co., San Francisco, CA. 17 Ziegler, E. I., H. Douglas, I. E. Sherman, C. E. Davis, and A. 1. Braude. 1973. Treatment of E. coli and Klebsiella bacteremia in agranulocytic animals with antiserum to UDP-GAL epimerase-deficient mutant. I. Immunol. 111:433.
The Ohio State University Ohio Agricultural Research and Development Genter Wooster 44691
ABSTRACT
New IMI caused by coliforms are highly associated with the 2-wk period prior to calving and early lactation (15). The high rate of coliform IMI at calving coincides with a period of reported immunosuppression (3, 10). An effective vaccine that would increase resistance to coliform infections during these periods would reduce significantly the losses caused by this disease. Considerable attention has been given to the protective effects of vaccinating laboratory animals and humans with the Rc rough mutant Escherichia coli (0111:B4) 15. Escherichia coli 15 lipopolysaccharide (LPS) lacks 0polysaccharide chains, thereby exposing the core antigens of LPS (4). The core antigens of LPS appear to be highly conserved among Gram-negative bacteria compared with the extremely heterogenous O-polysaccharide antigens. Both passive and active immunization with E. coli 15 protected laboratory animals against endotoxic shock and bacteremia by heterologous strains (2, 12, 17). Vaccinating dairy cows with E. coli 15 bacterin during the dry period and early lactation significantly reduced the incidence of clinical mastitis during the first 90 d of lactation (5). However, the investigators did not determine whether immunization reduced the incidence of IMI. The purpose of the current trial was to determine effects of an E. coli 15 bacterin on reducing naturally occurring IMI and clinical mastitis in a commercial dairy herd.
Eflficacy of an Escherichia coli (0111:B4) 15 bacterin for preventing naturally occurring IMI and clinical mastitis was tested in a 2.5-yr field trial in a 225~ow commercial herd. Cows with odd-numbered identification were vaccinated, and cows with evennumbered identification served as unvaccinated controls for each lactation during the study. Immunizations were subcutaneous on the upper part of the rib cage just posterior to the scapula at drying off, 30 d after drying off, and at calving. Percentage of quarters infected at calving with Gram-negative bacteria did not differ between treatment groups. A total of 67% of Gram-negative bacterial IMI present at calving in control cows became clinical during the first 90 d of lactation compared with 20% in vaccinated cows. Rate of Gram-negative bacterial clinical mastitis was higher in control cows than in vaccinated cows during the first 90 d of lactation. Immunization with the E. coli 15 bacterin did not reduce level of Gram-negative bacterial IMI at calving but did reduce incidence of clinical mastitis. (Key words: Escherichia coli, vaccine, mastitis) Abbreviation key: LPS
= lipopolysaccharide.
INTRODUCTION
MATERIALS AND METHODS
Gram-negative bacterial IMI are common causes of toxic clinical mastitis in dairy cattle.
Cooperating Herd
Received June 12, 1991. Accepted September 6, 1991. 1Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 99-91.
Experimental cows were in a commercial herd of approximately 225 lactating Holsteins. Lactating cows were in total confinement freestall housing. Free stalls were bedded with washed sand Cows were milked twice daily in a double-eight herringbone parlor. Milking hygiene included premilking and postmilking dis-
1992 J Dairy Sci 75:78-84
78
ESCHERICHIA COli 15 FIEID TRIAL
infection of teats with 5.25% sodium hypochlorite. All cows were dried off by abrupt cessation of milking, and all quarters were drytreated with an FDA approved dry cow therapy product. Dry cows were pastured with constant access to loose housing bedded with long straw. Two weeks prior to anticipated calving, cows were transferred to a 4.6- x 9.2-m maternity area and remained there until the fIrst regularly scheduled milking after parturition. The study was initiated in March 1988 and concluded in October 1990. Rolling herd averages were 8235 kg of milk and 307 kg of fat at the beginning of the study and 9600 kg of milk and 330 kg of fat at termination of the experiment. Rolling herd average SCC was 164,0001 00 of milk in March 1988 and 109,000/00 of milk in October 1990. Experimental Design
Cows entered the trial the day of drying off. Cows with odd-numbered identification were vaccinated, and cows with even-numbered identification served as unvaccinated controls for each lactation during the study. Escherichia coli bacterin was 5 00 of 109 cells of boiled E. coli J5/ml plus 1 00 of Freund's incomplete adjuvant (Difco Laboratories, Detroit, MI). Immunizations were at drying off, 30 dafter drying off, and within 24 h after parturition. Immunizations were subcutaneous on the upper part of the rib cage just posterior to the scapula. Milk samples
Bacteriological status of mammary quarters was determined by analyses of samples collected utilizing the following scheme. Duplicate quarter foremilk samples were collected aseptically by the herd manager from 1) all cows once during the 7- .05). between vaccinated cows and unvaccinated Rate of total clinical cases was .583/1000 controls (P > .05). Level of Gram-negative cow-days in parity 3 control cows and .095/ bacterial IMI was greater (P < .05) in control 1000 cow-days in parity 3 vaccinated cows cows than in vaccinated cows during yr 1 of (Table 3; P < .05). Gram-negative bacterial the study (2.1 and .4% quarters, respectively). Gram-negative bacterial IMI at calving in- clinical mastitis rate in parity >3 control cows creased during yr 2 to 2.3% of quarters com- was fourfold greater (P < .05) than the rate for pared with .4% during yr 1 in vaccinated cows. parity >3 vaccinated cows. Within lactation 1 Percentage of quarters infected with Gram- (Table 4), control cows had a greater (P < .05) negative bacteria at calving in unvaccinated rate of clinical Gram-negative bacterial masticows did not differ among the years. Percent- tis (.300/1000 cow-days) than vaccinated cows age of quarters infected with Gram-negative (.065/1000 cow-days). Rates of clinical Grambacteria at calving did not differ among parity negative bacterial mastitis did not differ begroups or between lactation 1 and 2 cows. tween lactation 1 and lactation 2 cows within Percentage distribution of species within experimental treatment groups. Both rates of total clinical cases and clinical Gram-negative bacterial IMI at calving in control cows was E. coli, 33.3%, Serratia species, Gram-negative cases were greater in control 33.3%; Proteus species, 25%; and Klebsiella cows than in vaccinated cows during yr 1 of species, 8.3%. Percentage distribution of spe- the study (Table 5). Rates of clinical mastitis cies within Gram-negative bacterial IMI at did not differ between treatment groups during calving in vaccinated cows was E. coli, 50%; yr 2 and 3. Rate of clinical Gram-negative Serratia species, 20%; Pseudomonas species, mastitis cases was greatest in yr 1 for control 20%; and Klebsiella species, 10%. A total of cows, but rates did not differ among years of 66.7% of Gram-negative bacterial IMI at calv- the study in vaccinated cows. In summary, 7 of (lactation 1 first lactation on the trial; lactation 2 = second and third lactations on the trial).
Journal of Dairy Science Vol. 75, No.1, 1992
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ESCHERICHIA COll 15 FIELD TRIAL
TABLE 2. Rate of clinical mastitis! in Escherichia coli 15 vaccinated (15) and unvaccinated cows (C) during a 2.5-yr field trial in a commercial dairy. First 90 d3 Bacteriological status
C
15
Gram-negative bacilli Environmental streptococci Staphylococcus species Actinomyces pyogenes Bacteriologically negative Total
.2558 .064 .021 .064 .106 .511
.047b .5708 .094.000 .023 .063 .023 .127 .141 .127 .328 .887
15
C
.140b
210 .070 .000 .351 .772
"~ates with differing superscripts within rows and comparisons differ (P < .05). 1Rate of clinical mastitis = number of clinical cases per 1000 cow-days. 2Rate per total lactation. 3Rate per f1I'St 90 d of lactation.
the 12 (58%) Gram~negative bacterial clinical cases in unvaccinated cows occurred during the first 90 d of lactation in parity >3 cows during the 1st yr of the study compared with 0 of 2 cases in vaccinated cows. Percentage distribution of species within Gram-negative bacterial clinical mastitis cases in control cows was E. coli, 50%; Serratia species, 25%; Klebsiella species, 16.7%; and Proteus species, 8.3%. Both Gram-negative bacterial clinical cases in vaccinated cows were E. coli. DISCUSSION
Vaccinating dairy cows with an E. coli J5 bacterin during the dry period and early lactation significantly reduced rate of Gramnegative bacterial clinical mastitis during this 2.5-yr study. These results agree with those of Gonzales et al. (5) in which incidence of clini-
cal mastitis was reduced by immunizing cows with the same vaccine during a 1.5-yr study in two California dairy herds. In addition, the magnitude of reduction was similar between trials. Rate of Gram-negative bacterial clinical mastitis was 4.1-fold lower in vaccinated cows versus controls in the present study compared with a 4.9-fold difference in the California trial. Data in the present trial suggest that protection offered by the vaccine was greatest for parity >3 cows in early lactation. TIlls corresponds with results of previous surveys showing that older cows during the first weeks following calving is the population at greatest risk to Gram-negative bacterial clinical mastitis (15). However, the data suggest that positive effects of vaccination were not restricted to older cows. Although differences were not significant, rates of Gram~negative bacterial clinical mastitis also tended to be higher in
TABLE 3. Rate of clinical mastitis! in Escherichia coli 15 vaccinated (15) and unvaccinated cows (C) during a 2.5-yr field trial in a commercial dairy by parity groups. Parity 2 Bacteriological status Gram-negative bacilli Environmental streptococci Staphylococcus species Actinomyces pyogenes Bacteriologically negative Total
C
.009 .198 .000 .000 .000 .280
Parity 3 15
Parity >3 15
C
.000 .194 .116 .097 .000.000 .000.000 .231 .291 .347 .5838
.000 .095 .000 .000 .000 .095 b
15
C 8
.388 .000 .038 .113 .075 .564
.085b .085 .043 .043 .171 .427
a,~tes with differing superscripts within rows and comparisons differ (P < .05). IRate of clinical mastitis = number of clinical cases per 1000 cow-days. lournal of Dairy Science Vol. 75, No.1, 1992
82
HOGAN ET AL.
TABLE 4. Rate of clinical mastitis l in Escherichia coli 15 vaccinated (15) and unvaccinated cows (C) during a 2.5-yr field ttial in a commercial dairy by number of laerations a cow was on the ttial. Lactation Bacteriological status Gnan-negative bacilli Environmental streptococci Staphylococcus species Actinomyces pyogenes Bacteriologically negative Total
Lactation
C
IS
.300"
.065 b
C
15
.155 .13O.()(l() .030.000 .032 .155 .129 .077 .389 .386
.099 .032 .030 .120 .570
.000 .000 .000 .000 .175 .175
a,~tes with differing superscripts within rows and comparisons differ (P < .05). IRate of clinical mastitis
= number of clinical
cases per 1000 cow-days.
2Rate among cows completing only one dry period on the ttial. 3Rate among cows completing second and third dry periods on the trial.
controls than in vaccinates in the other parity groups. Cows that underwent a second and third regimen of vaccination did not appear to have enhanced protection compared with those completing only one series of inununizations. This was evidenced by the fact that rates of clinical mastitis did not differ between lactation 1 and 2 vaccinated cows. Conclusions drawn from this study concerning the value of vaccinating cows in ensuing dry periods and lactations should be qualified by accounting for the relatively small number of animals completing more than one dry period. A total of 44 and 4 vaccinated cows completed two and three immunization regimens, respectively. A comparison of Gram-negative bacterial clinical cases within these 48 cows showed that one clinical
case occurred in cows during the first lactation on the trial compared with zero during second and subsequent lactations. Differences in rates of clinical mastitis between treatment groups changed during the trial. Vaccinated cows had a lower rate of Gram-negative bacterial clinical cases than controls during the first 12 mo, but differences between groups were not significant the last 18 mo. This apparent change in efficacy was not a result of decreased protection afforded by the vaccine because rates of clinical mastitis did not differ throughout the study in vaccinated cows. Rates of Gram-negative clinical mastitis in control cows were significantly reduced the last 18 mo of the study compared with the first 12 mo. The decrease in clinical mastitis was possibly caused by positive changes in one or
TABLE 5. Rate of clinical mastitis l in Escherichia coli 15 vaccinated (15) and unvaccinated cows (C) during a 2.5-yr field ttial in a commercial dairy by year of study. y~
1
2
3
Bacteriological status
C
15
C
IS
C
15
Gnan-negative bacilli Environmental streptococci Staphylococcus species Actinomyces pyogenes Bacteriologically negative Total
.430"' .000 .048 .048 .096 .621 a
.000b .000 .051 .000 .101 .152b
.124 .124 .000 .000 .083 .330
.095 .189 .000 .047 .047 .378
.000 .000 .000 1.104 .552 1.657
.000 .000 .000 .000 1.739 1.739
a,~tes with differing S1Iperscripts within rows and comparisons differ (P < .05). IRate of clinical mastitis number of clinical cases per 1000 cow-days.
=
2yr I = April
1988 to March 1989; yr 2 = April 1989 to March 1990; and yr 3 = April 1990 to September 1990.
Journal of Dairy Science Vol. 75, No.1, 1992
83
ESCHERICHIA COll 15 FIELD TRIAL
more management practices in the herd. Although care was taken by both herd managers and researchers conducting the trial to ensure that management practices other than experimental treatments did not differ between treatment groups, changes in husbandry practices were left to the discretion of the herd managers. These data illustrate the advantage of longer field trials to document the efficacy of vaccines as the incidence of naturally occurring infections and clinical cases changes within a herd. The advantages afforded by immunizing with the E. coli J5 bacterin will be minimized as environmental factors either reduce the exposure of cows to Gram-negative bacterial pathogens or enhance nonspecific host defense mechanisms. Immunization of cows with E. coli J5 during the dry period did not protect cows from IMI at parturition. Vaccination reduced the percentage of quarters infected at calving with Gram-negative bacteria the 1st yr but did not affect prevalence for the remainder of the study. Control and vaccinated cows also had similar percentage distributions of Gramnegative bacterial species within IMI diagnosed at calving. The most striking difference between treatment groups was that 66.7% of IMI in controls became clinical during early lactation compared with 20% in vaccinates. These data imply that the E. coli J5 vaccine did not prevent the occurrence of IMI but did reduce the severity of the disease. The mechanism of action by which immunization with E. coli J5 provides protection appears to be related to increased antibody to the highly conserved LPS core antigens. Crossreactivity of antisera from vaccinated animals to heterologous smooth Gram-negative bacteria was due to antibody directed against core antigens (1, 13, 14). The protection afforded by vaccinating with E. coli J5 has been hypothesized as being due to increased opsonization of heterologous bacteria and detoxification of LPS by antibody (2, 17). Although serological testing was not within the practical limitations of this study, an earlier study showed that cows vaccinated with the bacterin had increased serum and milk: titers of IgM and opsonic activity against a heterologous E. coli strain (9). The relevance of increased IgM concentration in decreasing mastitis is questionable because milk from unvaccinated cows
has sufficient opsonic actlVity (7). Experimental challenge trials revealed that vaccination did not prevent IMI, and antibody titer response to immunization was minimal prior to infection (6). However, serum IgG titer increased more in vaccinated cows than in unvaccinated controls 6 wk following experimental challenge. Mastitis data in the current and previous studies concur with experiments in laboratory animals that suggest the primary protective action of the E. coli J5 vaccine is the reduction of systemic responses from endotoxemia following Gram-negative bacterial infections, thus reducing the severity of the disease. ACKNOWLEDGMENTS
The authors thank Jim Collor, University of California-Davis, for kindly providing the antigen; the Ayers Farm, Inc. for its unselfish cooperation; and the Ohio Dairy Farmers' Federation for partial financial support of this experiment. REFERENCES 1 Baumgartner, I. D., T. X. O'Brien, T. N. Kirkland, M P. Glauser, and B. I. Ziegler. 1987. Demonstration of cross-reactive antibodies to smooth Gram-negative bacteria in antiserum to Escherichia coli 15 J. Infecl Dis. 156:136. 2 Braude, A. I., E. J. Ziegler, H. Douglas, and J. A. McCutchan. 1977. Antibody to cell wall glycolipid of Gram-negative bacteria: induction of immunity to bacteremia and endotoxemia. I. Infect. Dis. 136:S167. 3 Craven, N., and M. R. Williams. 1985. Defenses of the bovine mammary gland against infection and prospects for their enhancements. Vet. Immunol. Immunopathol. 2:71. 4 ElOOm, A. D., and E. C. Heath. 1965. The biosynthesis of cell wall lipopolysaccharide in Escherichia coli. J. Biol. Chern.. 5:1919. 5 Gonzales, R. N., J. S. Cullor, D. B. 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. Vel Res. 53:301. 6 Hill, A. W. 1991. Vaccination of cows with rough Escherichia coli mutants fails to protect against experimental intramammary bacterial challenge. Vel Res. Common. 15:7. 7 Hill, A. W., DJ.S. Heneghan, and M. R. Williams. 1983. The opsonic activity of bovine milk whey for the phagocytosis and killing by neutrophils of encapsulated and non-encapsulated Escherichia coli. Vet. Microbiol. 8:293. 8 Hogan, I. S., K. L. Smitb. K. H. Hoblet, P. S. Schoenberger, D. A. Todhunter, W. D. Hueston, D. B. PritchJournal of Dairy Science Vol. 75, No.1, 1992
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ard, G. L. Bowman, L. E. Heider, B. L. Brockett, and H. R. Conrad. 1989. Field survey of mastitis in low somatic cell count herds. I. Dairy Sci. 72:1547. 9 Hogan, I. S., D. A. Todhunter, G. M. Tomita, K. L. Smith, and P. S. Schoenberger. 1992. Opsonic activity of bovine serum and mammary secretion after Escherichia coli 15 vaccination. I. Dairy Sci. 75:12. 10 Kehrli, M. E., B. I. Nonnecke, and I. A. Roth. 1989. Alterations in bovine neutrophil function during the periparturient period. Am. I. Vet. Res. 50:207. 11 Koneman, E. W., S. D. Allen, V. R. Dowell, and H. M. Sommer. 1983. The enterobacteriaceae. Page 57 in Color atlas and textbook of diagnostic microbiology. 2nd ed. I. B. Lippincott Co., New York, NY. 12 Marks, M. I., E. I. Ziegler, H. Douglas, L. B. Corbeil, and A. I. Braude. 1982. Induction of immunity against lethal Haemophilus influenzae type b infection by Escherichia coli core lipopolysaccharide. I. CIin. Invest. 69:742.
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13 McCallus, D. E., and N. L. Norcross. 1987. Antibody specific for Escherichia coli 15 cross-reacts to various degrees with an Escherichia coli clinical isolate grown for different lengths of time. Infect. Immun. 55:1042. 14 Mulharia, L. M., G. Crockford, W. C. Bogard, and R.E.W. Hancock. 1984. Monoclonal antibodies specific for Escherichia coli 15 lipopolysaccharide: crossreaction with other Gram-negative bacterial species. Infect. Immun. 45:631. 15 Smith, K. L., D. A. Todhunter, and P. S. Schoenberger. 1985. Environmental mastitis: cause, prevalence, prevention. I. Dairy Sci. 68:1531. 16 Sokal, R. R., and F. I. Rohlf. 1981. Biometry. 2nd ed. W. H. Freeman and Co., San Francisco, CA. 17 Ziegler, E. I., H. Douglas, I. E. Sherman, C. E. Davis, and A. 1. Braude. 1973. Treatment of E. coli and Klebsiella bacteremia in agranulocytic animals with antiserum to UDP-GAL epimerase-deficient mutant. I. Immunol. 111:433.
