J. S. Hogan 1
K. L. Smith
P. Schoenberger
S. Romig
L. Thompson
The Ohio State University
Department of Animal Sciences
1 For more information, contact at: The Ohio State University, Ohio Agricultural Research and Development Center, 302 Pounden Hall, 1680 Madison Avenue, Wooster, OH 44691; 330-263-3801; e-mail: hogan.4@osu.edu
The effect of immunization schedule on responses of antibody titers was tested following vaccination with an Escherichia coli J5 bacterium. Eighteen cows were equally distributed among three immunization schedules: (1) subcutaneous injection at 14 days prior to the end of lactation, intramammary immunization at seven days after drying off, and subcutaneous injection at 30 days into the dry period; (2) subcutaneous injections at drying off, at 30 days into the dry period, and within 12 hours after calving; and (3) unimmunized controls. Intramammary immunization increased rectal temperatures at 12 hours after infusion compared with unimmunized controls. Subcutaneous injections did not induce febrile responses. Intramammary immunization enhanced immunoglobulin G (IgG) titers in serum and whey on day zero of lactation compared with subcutaneous immunizations. Serum IgG titers also were greater at day 30 of the dry period and at days 14 and 21 of lactation in cows that received intramammary immunization compared with cows that were vaccinated by subcutaneous injections only. Immunoglobulin M (IgM) titers in whey and serum on day 21 of lactation were greater in cows that received intramammary immunizations compared with cows that were immunized by subcutaneous injection only.
The use of Escherichia coli J5 bacterins during the dry period has reduced the incidence and severity of clinical coliform mastitis (Gonzales et al., 1989). The protection afforded by the use of E. coli J5 vaccines has been ascribed to enhanced concentrations of IgG and IgM in serum and mammary secretion directed toward conserved antigens on the cellular surface of Gram-negative bacteria. The role of IgG in protecting immunized cows has been attributed to the toxin-neutralizing abilities of IgG binding to the core antigens of lipopolysaccharide (Tyler et al., 1992). Cows immunized with E. coli J5 had enhanced IgM titers that were correlated with increased opsonization and phagocytosis of heterologous E. coli by bovine neutrophils (Hogan et al., 1992).
Vaccination schedules in E. coli J5 efficacy trials have involved systemic immunizations during late lactation, the dry period, and the periparturient period. Australian researchers (Lascelles and McDowell, 1974; Watson and Lascelles, 1975) reported local immunization of ruminant mammary glands with bacterial antigens during the dry period induced local production of immunoglobulin A (IgA) and IgM persisting from parturition into early lactation. Saif et al. (1984) also reported that local immunization of bovine mammary glands during the early dry period resulted in enhanced IgG titers directed against viral antigens. The purpose of the current trial was to compare IgG and IgM titers of cows vaccinated with a conventional E. coli J5 systemic immunization schedule with those of cows vaccinated with a schedule including intramammary immunization during the early dry period.
Eighteen cows were randomly assigned to one of the following E. coli J5 immunization schedules: (1) subcutaneous injection at 14 days prior to end of lactation, intramammary immunization at seven days after drying off, and subcutaneous injection at 30 days into the dry period; (2) subcutaneous injections at drying off, at 30 days into the dry period, and within 12 hours after calving (conventional schedule); and (3) unimmunized controls. Subcutaneous injections were administered on the upper part of the rib cage posterior to the scapula. Intramammary immunizations of 2.5 ml of vaccine were infused by means of the teat canal into each of the four mammary glands using a 34-mm cannula (Jorgensen Laboratories, Inc., Loveland, Colo.). The E. coli J5 bacterin consisted of 5 ml of 109 boiled cells/ml emulsified with 5 ml of Freund's incomplete adjuvant.
All cows were dried off by abrupt cessation of milking at approximately 60 days prior to anticipated calving, and all four quarters were dry treated with an antibiotic intramammary infusion product (Tomorrow®; Franklin Laboratories, Amarillo, Tex.) the last milking of lactation. Experimental cows were housed and managed similarly.
Rectal temperatures were measured 0, 12, 24, and 48 hours after each immunization. Rectal temperatures of control cows were measured at time points corresponding to those of immunized cows on day 14 prior to drying off, the day of drying off, days seven and 30 of the dry period, and day zero of lactation. An ELISA was used to determine antibody titer in serum and whey to whole cell E. coli J5 (Tyler et al., 1988).
Intramammary immunization increased rectal temperatures at 12 hours after infusion compared with unimmunized controls (Figure 1). On day seven of the dry period, average rectal temperature was 104.5ºF at 12 hours following intramammary immunization compared with 101.8ºF for unimmunized cows at the same time. Average rectal temperature had returned to 102.7ºF and 102.2ºF by 24 and 48 hours after intramammary infusion, respectively. Subcutaneous injections did not induce febrile responses.
Vaccination with E. coli J5 increased IgG titers in mammary secretion at calving and early lactation. Immunization increased IgG titers against whole cell E. coli J5 in mammary secretions collected at calving and days seven and 21 of lactation compared with mammary secretion from unimmunized control cows. In addition, the immunization schedule affected IgG titers in mammary secretion at calving. Average IgG titers in mammary secretion at calving from cows receiving intramammary immunization was sixfold higher compared with cows receiving subcutaneous immunizations only. Immunoglobulin G titers in mammary secretion at days seven, 14, and 21 of lactation did not differ between cows treated with the two different immunizations protocols.
Immunoglobulin M titers in mammary secretion directed against E. coli J5 (Figure 3) did not differ between vaccinated and unvaccinated controls at calving and early lactation. In comparisons between vaccination schedules, IgM titers in mammary secretion were higher at day 21 of lactation in cows receiving intramammary immunization compared with those of cows immunized by subcutaneous injections only. Immunoglobulin M titers in mammary secretion at calving and on days seven and 14 of lactation did not differ between immunization schedules.
Immunization increased IgG titers in serum on days 14 and 21 of lactation compared with unimmunized cows. However, serum IgG titers did not differ between vaccinated and unvaccinated cows on day 30 of the dry period, at calving, or day seven of lactation. Within vaccinated cows, immunization schedule did affect serum IgG titers. Cows vaccinated by intramammary immunization had higher serum IgG titers on day 30 of the dry period, at calving, and days 14 and 21 of lactation than did cows receiving subcutaneous immunizations only.
Serum IgM titers against E. coli J5 (Figure 5) were higher in vaccinated cows than unvaccinated cows at calving. However, immunization did not affect IgM titers in serum at day 30 of the dry period or during early lactation. Cows receiving intramammary immunization had higher IgM titers on day 21 of lactation compared with cows immunized by subcutaneous injections only. Other differences among IgM titers in serum within sampling periods were not significant.
An E. coli J5 vaccination schedule that included an intramammary immunization sequenced between two subcutaneous injections resulted in greater responses of IgG and IgM titers than did a vaccination protocol involving three systemic immunizations. These results support the findings of Watson and Lascelles (1975) that local infusions of antigen into the involuting mammary gland can enhance antibody responses when given as a booster following systemic immunization. The hypothesis proposed by these researchers (Watson and Lascelles, 1975) was that peripheral antigenic stimulation resulted in seeding of a proportion of lymphoid cells into mammary tissue that was sufficient to give rise to local antibody production. The antibody isotypes produced locally in the mammary gland included IgM and IgA, but not IgG.
Both IgM and IgG titers in mammary secretions were elevated in intramammarily immunized cows compared with conventionally immunized cows in the current study. The enhanced IgM titers in intramammarily infused cows compared with conventionally immunized cows were detected only on day 21 of lactation. Serum IgM titers were increased in intramammarily immunized cows compared with those from conventionally vaccinated cows on day 21 of lactation. This serum increase in IgM corresponded with the increase in milk IgM titers. Although the results of the current trial do not discount the possibility of local IgM production, the similarities between IgM titer responses in milk and serum of intramammarily immunized cows suggest the passive transfer of IgM from blood to milk.
Mammary secretion of IgG titers was elevated only at calving. Bovine IgG is actively transported from blood to mammary secretion (Lascelles and McDowell, 1974). Serum IgG titers also were elevated in intramammarily immunized cows compared with conventionally immunized cows in the current trial. These results support the suggestions by Saif et al. (1984) that an intramammary booster following systemic immunization with viral antigens could elicit an enhanced systemic antibody response in addition to local immunity. Serum IgG titers were enhanced in intramammarily immunized cows compared with conventionally immunized cows from 30 days into the dry period through early lactation in the current trial. The elevated mammary secretion and serum IgG titers at calving suggested that intramammary immunization elicited a greater systemic immune response than did subcutaneous injections alone. The bovine mammary gland has numerous lymphocytes infiltrating into stroma and epithelium after seven days of involution. Cells stimulated locally in the gland following an intramammary booster may traffic to local lymphatic tissues, bolstering systemic antibody responses.
The relationship between IgG titers in serum and mammary secretion in intramammarily immunized cows can possibly be explained by the selective transfer of IgG1 across secretory cells. Although selective transfer of IgG1 continues through lactation, the relative magnitude of the transfer was considerably less during lactation than during colostrum formation (Lascelles and McDowell, 1974). Therefore, the observations in the current trial that serum IgG titers remained elevated in intramammarily immunized cows while milk IgG titers were comparable between vaccination groups are consistent with the previously reported decrease in selective transfer of IgG as mammary secretion changes from colostrum to milk.
The E. coli J5 vaccination schedule that included intramammary immunization enhanced antibody titer responses; however, one possible disadvantage of this scheme was the increased rectal temperature at 12 hours after intramammary immunization. Although this febrile response was transitory and had no observable negative impact on the health of cows, care should be exercised when using pyrogenic antigens. An alternative may be the use of detoxified lipopolysaccharide that has been shown to be immunogenic to dairy cows but did not induce fever when infused into a lactating mammary gland. The enhanced IgM and IgG titers in both serum and mammary secretions after intramammary immunization may have practical implications for maximizing protection afforded by E. coli J5 bacterins.
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