Efficacy of an Escherichia coli J5 Mastitis Vaccine in an Experimental Challenge Trial1

Efficacy of an Escherichia coli J5 Mastitis Vaccine in an Experimental Challenge TriaJ1 J. S. HOGAN, W. P. WEISS, D. A. TODHUNTER, K. L. SMITH, and P. S. SCHOENBERGER Department of Dalry Science The Ohio State University Ohio Agricultural Research and Development Center Wooster 44691 ABSTRACT

Abbreviation key: LPS = lipopolysaccharide, PBS = phosphate-buffered saline, TSB = trypticase soy broth.

An Escherichia coli (0111:B4) J5 bacterin was tested for efficacy in reducing IMI and severity of clinical coliform mastitis in an experimental challenge trial. Ten cows were immunized at drying off, 30 d after drying off, and at calving. Ten control cows were not immunized. Right front quarters of all cows were infused with a heterologous strain of E. coli approximately 30 d after calving. Vaccinated cows had lower bacterial counts in milk and lower rectal temperatures than unvaccinated controls following intramammary challenge. Milk production and DM! were more depressed in controls than in vaccinated cows. Milk SCC did not differ between experimental groups. Mean serum IgG titer to whole cell E. coli J5 was significantly greater in vaccinated than in unvaccinated cows at 30 d after drying off, day of challenge, and 7 d postchallenge. Milk IgG titer to E. coli J5 was higher at challenge in vaccinated than in control cows. Vaccination with the E. coli J5 bacterin did not prevent IMI but did reduce severity of clinical signs following intramammary experimental challenge with a heterologous E. coli strain. (Key words: Escherichia coli, vaccine, mastitis) .

INTRODUCTION

Received June 17, 1991. Accepted September 6, 1991. lSalaries and research support were provided by slate and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 121-91. 1992 J Dairy Sci 75:415-422

A series of controlled field trials have shown that parenteral immunization of dairy cows with Escherichia coli (Ol11:B4) J5 bacterin significantly reduced rate of clinical Gram-negative bacterial (2, 5). Results of one field trial revealed that prevalence of IMI was not affected by E. coli J5 immunization (9), although incidence of clinical mastitis was reduced. The greatest difference between treatment groups was that 66.7% of IMI at calving in unvaccinated cows became clinical during early lactation compared with 20% in vaccinated cows. These data suggest that vaccination provided little prophylactic benefit against naturally occurring IMI but did reduce the severity of disease once IMI were established. The mechanisms by which E. coli J5 vaccines provide protection have been investigated in trials in which laboratory animals were experimentally infected with Gramnegative bacteria. Active immunization enhanced resistance against experimentally induced infections by a heterogenous group of Gram-negative bacteria, including the coliforms, Pseudomonas species, and HaemophiIus injluenzae (1, 12). Conclusions were that vaccination effectively reduced incidence of infections and mitigated severity of clinical signs in infected animals. Dairy cows vaccinated with an E. coli J5 bacterin were not protected against experimental IMI by a heterologous E. coli strain (7), despite an enhanced antibody response prior to challenge. Reasons for failure of the E. coli J5 vaccine to provide protection against experimental IMI and clinical mastitis when the vaccine was effective

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under natural exposure conditions are unknown and warrant further investigation. The purpose of the current trial was to detennine effects of an E. coli J5 bacterin on reducing experimentally induced IMI and clinical mastitis in early lactation dairy cows. MATERIALS AND METHODS Experimental Animals

Twenty cows in the Ohio Agricultural Research and Development Center dairy herd were assigned to two groups of 10 cows. Cows in one group were vaccinated with an E. coli J5 bacterin. All vaccinated cows were immunized at drying off, 30 d after drying off, and within 48 h after calving. hnmunizations were subcutaneous on the upper part of the rib cage just posterior to the scapula. The E. coli bacterin was 2 m1 of 1()9 cells/ml of fonnalinkilled E. coli J-5 in oil adjuvant (Fort Dodge Laboratories, Fort Dodge, IA). Cows in the other group were unvaccinated controls. Breed distributions were 6 Holstein and 4 Jersey in the vaccinated group and 7 Holstein and 3 Jersey in the control group. Parities averaged 3 among control cows and 3.1 among vaccinated cows. Cows in both groups were dried off by abmpt cessation of milking, and all four quarters were dry-treated with 300 mg of cephapirin benzathine product (fomorrow, Franklin Laboratories, Amarillo, TX) 60 d prior to anticipated calving. Cows in both treatment groups were housed and managed similarly. Following parturition, all cows were fed the same diet. which was adequate in all nutrients, including .3 ppm of selenium and 1000 mg of vitamin E. Blood was collected from each cow immediately prior to bacterial challenge to determine vitamin E (10) and glutathione peroxidase status (16). Intramammary Challenge

Escherichia coli 487, a smooth strain originally isolated from a cow with clinical mastitis, was used as the intramammaIy challenge strain. Challenge inoculum was prepared by inoculating a frozen stock culture of E. coli 487 into trypticase soy broth (TSB). The TSB culture was incubated for 18 h at 37°C in a gyratory incubator at 100 rpm. A total of .01 Journal of Dairy Science Vol. 75. No.2. 1992

ml of this culture was inoculated into fresh TSB and incubated 2.5 h at 37°C and 100 rpm. The log phase TSB broth culture was centrifuged, and the pellet was resuspended into 5 ml of phosphate-bufIered saline (PBS). A 1:10 dilution of culture was made in PBS and adjusted to 67.5% transmission at 540 om (Beckman DU-50 Spectrophotometer, Beckman Instruments, Fullerton, CA). Six additional serial 10-fold dilutions in PBS were made, and colony-fonning units per milliliter were determined by plating 1 ml in duplicate in McConkey agar pour plates. The right front mammary quarter of each control cow and each immunized cow was challenged by infusion of approximately 30 cfu of E. coli 487 suspended in 1 ml of PBS. Cows were challenged approximately 30 d (X = 28.6 d; range = 23 to 32 d) into lactation. Infusions were 2 h after evening milking. Only uninfected quarters were infused. Therapy for peracute clinical signs was limited to oral fluids beginning 24 h after bacterial challenge. Quarter Foremilk Samples

Incidence of naturally occurring IMI at drying off and during early lactation was determined by foremilk samples taken 14 and 7 d prior to drying off, the day of drying off, and d 0, 3, 7, 14, and 21 of lactation. Quarter foremilk samples also were collected the day prior to bacterial challenge, immediately prior to challenge, 6, 12, 15, 18, 21, and 23 h postchallenge, and d 2,3,4, and 7 after challenge. Sample collection and microbiological procedures were described by Smith et al. (18). All Gram-negative isolates were identified by the API-20E (Analytab Products, Plainview, NY) system. The colony-fonning units per milliliter and SCC were determined in quarter foremilk samples during the postchallenge period. Colonyfonning units were determined by appropriate lo-fold dilutions of sample in PBS. The initial inocula were duplicate 1-ml pour plates of undiluted milk in McConkey agar. Dilutions were plated on the swface of McConkey agar plates. All dilutions were in duplicate. Somatic cell counts per milliliter of milk were determined by Coulter Counter (Coulter Electronics, Hialeah, FL). Samples from clinical quarters were diluted 1:10 (milk:PBS) for counting.

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Data were expressed as 10g1O cfu/mI and 10g1O SCC/mI. Clinical status of all quarters was recorded at the time that quarter foremilk samples were obtained. Clinical status was recorded on a scale from 1 to 5: 1 = nonnal milk and nonnal quarter; 2 = nonnal quarter but questionable milk; 3 = nonnal quarter but abnonnal milk; 4 = a swollen quarter and abnonnal milk; and 5 = swollen quarter, abnonnal milk, and systemic signs of infection Body Temperature

Rectal temperature was determined 0, 12, 24, 48, and 72 h following all immunizations. Rectal temperatures were measured immediately prior to challenge and at each time that quarter foremilk samples were collected postchallenge.

Statistical Analyses

Treatment differences between bacterial count, SCC, rectal temperature, DMI, and milk production means were tested by least squares ANOVA. Antibody titer data were tested for nonnality by the Shapiro-Wilkes procedure (17). Treatment differences were tested by least squares analysis of covariance; the titer at drying off was the covariant. Relationships between antibody titers at challenge and peak bacterial counts in milk were measured by Pearson's correlation coefficients (17). RESULTS Rectaltemperatures~n~roan~within

72 h after immunization at either drying off, 30 d after drying off, or at calving. No swelling or lumps were observed at injection sites. Means and standard errors for plasma vitamin E concentration were 2.4 ± .3 ~g/mI in vaccinated Milk Production and OMI cows and 2.9 ± .5 ~g/mI in control cows (P > Milk production was measured electroni- .05). Means and standard errors for whole cally at each milking for all cows. Daily feed blood glutathione peroxidase concentrations intake was recorded for all cows. Post- also did not differ between groups (control challenge daily milk production and DMI were cows 76 ± 3 vs. vaccinated cows 80 ± 5 expressed as percentages of average values for millienzyme units/mg of hemoglobin). The the 7 d prior to challenge [(b/a), where a = only naturally occurring Gram-negative bacteraverage value for the 7 d prior to challenge and ial IMI diagnosed was an Enterobacter cloab = daily value postchallenge]. cae infection present at calving that persisted to d 14 of lactation without clinical signs in a Antibody Titer vaccinated cow. The IMI was spontaneously eliminated without antibiotic therapy. An ELISA procedure was used to detennine Means and standard errors in colonyantibody titers in serum and mammary secretions to E. coli J5 and E. coli 487. Sera and forming units infused into mammary quarters mammary secretions were collected at drying were 35 ± 5 in control cows compared with 32 off, at calving, d 21 of lactation, the day of ± 4 in vaccinated cows. Bacterial counts were bacterial challenge, and 7 d postehallenge. Se- lower (P < .05) in vaccinated cows than in rum samples also were collected 30 d after control cows at 12, 15, 18, 21, and 23 h drying off. Escherichia coli J5 were incubated postchallenge (Figure 1). Neither speed of re18 h at 37°C. Escherichia coli 487 were in- sponse nor maximal sec differed between cubated for 2.5 h under the same culture condi- groups (Figure 2). Rectal temperatures were tions described for the experimental challenge. higher (P < .05) in control cows than in vacciThe ELISA procedures were essentially those nated cows at 12 and 15 h postchallenge detailed by Tyler et al. (20). Heat-killed bacte- (Figure 3). Rectal body temperature exceeded ria were coated into microtiter wells by incu- 4O.1°C for each cow following bacterial infubation overnight at 37°C. Isotypes were deter- sion. Nine of 10 cows (90%) in both experimined by rabbit anti-bovine IgG (Sigma mental groups were diagnosed as clinical code Chemical Company, St. Louis, MO) and goat 5 by 18 h postchallenge. anti-bovine IgM (Kirkegaard and Perry LaboMilk production was depressed more (P < ratories, Gaithersburg, MD). Titer data were .05) in control cows than in vaccinated cows d expressed as the reciprocal of the dilution log2. 3, 4, 5, and 6 postchallenge (Figure 4). Dry Journal of Dairy Science Vol. 75, No.2, 1992

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Serum IgG antibody titer to E. coli 15 was greater in vaccinated cows than in controls at d 30 of the dry period, at time of intramammary challenge, and 7 d postchallenge (P < .05; Figure 6). Milk IgG antibody titer to E. coli 15 was greater in vaccinated cows than in controls at time of bacterial challenge (P < .05; Figure 7) but did not differ between treatments at any other sample period. Serum and mammary secretion IgM titers to E. coli 15 did not differ between treatment groups. Serum and mammary secretion IgG and IgM titers to E. coli

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DISCUSSION

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Escherichia coli J5 in immunized cows with E. coli J5 and in unimmunized controls cballenged by intramammary infusion with E. coli 487. Samples were collected at drying off (D - 0), at calving (C + 0), 21 d into lactation (C + 21), immediately prior to bacterial cballenge (C + 30), and 7 d posteballenge (C + 37). Values are covariantadjusted least squares means of 10 cows. Bars show standard errors. Journal of Dairy Science Vol. 75, No.2, 1992

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Figure 9. Mammary secretion antibody titer (Anti) to Escherichia coli 487 in cows immunized with E. coli IS and in unimmunized controls cbal1enged by intramammary infusion with E. coli 487. Samples were conected at drying off (D - 0), at calving (C + 0), 21 d into lactation (C + 21), immediately prior to bacterial challenge (C + 30), and 7 d posteballenge (C + 37). Values are oovariantadjusted least squares means of 10 cows. Bars show standard errors.

during clinical colifonn mastitis result from bacterial release of lipopolysaccharide (LPS). To reduce the severity of acute clinical mastitis either bacterial growth must be inhibited or the effects of LPS neutralized (3). Results of the current study suggest that decreased clinical signs in immunized cows was from suppressed bacterial growth in milk. Both serum and milk total IgG titers to E. coli J5 at time of challenge were negatively correlated with peak bacterial counts in milk during experimental IMI. Bacterial growth and IgM titers to E. coli 487 were not related. Whether the relationship between enhanced IgG titers and lower bacterial counts was related to opsonization of bacteria and clearance by phagocytes or to a bacteriostatic system is unknown. Active immunization of farm animals with Rc rough mutants has been theorized to decrease severity of disease by enhancing clearance of Gram-negative bacteria and LPS or by masking active LPS epitopes (21). Serum IgGl titers to LPS core antigens in adult cows were nonnally distributed and negatively related to risk. of naturally

occurring colifonn clinical mastitis (20). However, data are limited that show decidedly that E. coli J5 vaccination enhances neutralization of LPS released from Gram-negative bacterial IMI. Escherichia coli J5 intramammary immunization had no protective effect against clinical signs following sterile LPS intramammary infusion (19). Vaccination did not prevent experimentally induced colifonn IML Previous natural experimental challenge and natural exposure trials also have shown that use of E. coli J5 vaccines did not prevent IMI (7, 9). The ability of a cow to defend against E. coli IMI appears to be related to the speed with which neutrophils can be mobilized from blood into the gland and to the concentration of opsonin present in mammary secretion (8). The primary opsonin of Gram-negative bacteria in milk is IgM (6). Immunization with an E. coli J5 bacterin resulted in increased serum and milk IgM, which correlated with increased opsonization of E. coli 487 (11). Vaccination did not affect IgM titer to E. coli J5 and E. coli 487 in either

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serum or milk in the present study. McCabe et al. (13) proposed that immunization with rough Gram-negative bacteria resulted in increased IgM that is involved in bactericidal activity but has little effect once an infection has become established. Decreases in endotoxic shock and mortality were related to increased IgG concentrations and detoxification of LPS. The principal immune response resulting from immunization in the present study was an increase in IgG titer. The mechanism of action by which immunization with E. coli ]5 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 (14, 15). McCallus and Norcross (14) reported that antisera to E. coli ]5 crossreacted with a heterologous E. coli greatest during log growth of bacteria when O-polysaccharide side chain formation was incomplete, thus exposing core antigens. Escherichia coli 487 cultures used to inoculate mammary glands and used as whole cell antigen in titer assays were log phase cultures. However, milk and serum titers to E. coli 487 showed minimal enhancement in vaccinated cows. Other studies have shown positive effects of passive and active E. coli ]5 immunization; minimal supportive data demonstrated enhanced antibody crossreactivity to other bacterial strains (22). The lack of demonstrated crossreactivity may be due to low specificity of antigen-antibody detection assays. Little is known conceming which Ig are protective and concerning the exact epitopes to which Ig must bind to protect animals from the deleterious effects of Gram-negative bacterial infections (4).

CONCLUSIONS Management practices that diminish clinical mastitis are extremely valuable in many dairy herds. Previous surveys have shown that 81% of Gram-negative bacterial IMI become clinical during early lactation and that Gramnegative bacterial clinical cases account for 32% of total and 60% of peracute clinical cases (18). With the exception of SCC, each quantitative measure of clinical signs was mitigated in vaccinated cows compared with

those measures in controls in the present study. The current and prior studies (2, 5, 9) suggest that vaccinating cows during the dry period and early lactation with E. coli ]5 may be a practical management tool to reduce the incidence and severity of clinical Gram-negative bacterial mastitis.

REFERENCES 1 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 en
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Salmonella minnesota: protective activity of IgM and to the R595 (Re chemotype) mutant. 1. Infect. Dis. 158:29l. 14 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. 15 Mutharia. 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. 16 Pagalia, D. E., and W. N. Valentine. 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Coo. Moo. 70:158. 17 SAS® User's Guide: Statistics, Version 5 Edition. 1985. SAS lust., Inc., Cary, NC.

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18 Smith, K. L., D. A. Todhunter, and P. S. Schoenberger. 1985. Environmental mastitis: cause, prevalence, prevention. J. Dairy Sci. 68:1531. 19 Todhunter, D. A., K. L. Smith, and P. S. Schoenberger. 1985. Response to intramammary challenge with endotoxin in cows immunized with Escherichia coli (15 mutant). 1. Dairy Sci. 68(Suppl. 1):204.(Abstr.) 20 Tyler, 1. W.,I. S. Cullor, B. 1. Osburn, R. B. Bushnell, and B. W. Fenwick. 1988. Relationship between serologic recognition of Escherichia coli 0111:B4 (IS) and clinical coliform mastitis. Am. J. Vet. Res. 49: 1950. 21 Tyler, 1. W., 1. S. Cullor, S. J. Spier, and B. P. Smith. 1990. Immunity targeting common core antigens of gram-negative bacteria. 1. Vet. Intern. Moo. 4:17. 22 Ziegler, E. J. 1988. Protective antibody to endotoxin core: the Emperor's new clothes? J. Infect. Dis. 158: 286.